molecular dissection of g-protein coupled receptor ...€¦ · figure ii.1: sequence weighting...

146
MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR SIGNALING AND OLIGOMERIZATION BY MICHAEL RIZZO A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Biology December, 2019 Winston-Salem, North Carolina Approved By: Erik C. Johnson, Ph.D. Advisor Wayne E. Pratt, Ph.D. Chair Pat C. Lord, Ph.D. Gloria K. Muday, Ph.D. Ke Zhang, Ph.D.

Upload: others

Post on 08-Oct-2020

1 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR SIGNALING AND OLIGOMERIZATION

BY

MICHAEL RIZZO

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Biology

December, 2019

Winston-Salem, North Carolina

Approved By:

Erik C. Johnson, Ph.D. Advisor

Wayne E. Pratt, Ph.D. Chair

Pat C. Lord, Ph.D.

Gloria K. Muday, Ph.D.

Ke Zhang, Ph.D.

Page 2: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

ii

ACKNOWLEDGEMENTS

I would first like to thank my advisor, Dr. Erik Johnson, for his support, expertise,

and leadership during my time in his lab. Without him, the work herein would not be

possible. I would also like to thank the members of my committee, Dr. Gloria Muday, Dr.

Ke Zhang, Dr. Wayne Pratt, and Dr. Pat Lord, for their guidance and advice that helped

improve the quality of the research presented here.

I would also like to thank members of the Johnson lab, both past and present, for

being valuable colleagues and friends. I would especially like to thank Dr. Jason Braco,

Dr. Jon Fisher, Dr. Jake Saunders, and Becky Perry, all of whom spent a great deal of

time offering me advice, proofreading grants and manuscripts, and overall supporting me

through the ups and downs of the research process.

Finally, I would like to thank my family, both for instilling in me a passion for

knowledge and education, and for their continued support. In particular, I would like to

thank my wife Emerald – I am forever indebted to you for your support throughout this

process, and I will never forget the sacrifices you made to help me get to where I am

today.

Page 3: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………………ii

TABLE OF CONTENTS………………………………………………………………...iii

LIST OF ABBREVIATIONS…………………………………………………………….v

LIST OF TABLES………………………………………………………………………..x

LIST OF FIGURES………………………………………………………………………xi

ABSTRACT……………………………………………………………………………..xii

CHAPTER I: G-protein coupled receptors– A review of structure-function relationships

critical for receptor signaling ……………………………………..………………………1

REFERENCES…………………………………………………………………..37

CHAPTER II: Unexpected role of a conserved domain in extracellular loop 1 in G

protein coupled receptor trafficking……………………………………………………...56

ABSTRACT……………………………………………………………………...57

INTRODUCTION……………………………………………………………….58

METHODS………………………………………………………………………60

RESULTS………………………………………………………………………..63

DISCUSSION……………………………………………………………………68

REFERENCES…………………………………………………………………..72

Page 4: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

iv

CHAPTER III: Homodimerization of Drosophila Class A neuropeptide GPCRs:

Evidence for conservation of GPCR dimerization throughout metazoan evolution…….89

ABSTRACT……………………………………………………………………..90

INTRODUCTION……………………………………………………………….91

METHODS………………………………………………………………………97

RESULTS………………………………………………………………………100

DISCUSSION…………………………………………………………………..104

REFERENCES…………………………………………………………………108

CHAPTER IV: Conclusions and future directions……………….…………………….125

REFERENCES…………………………………………………………………129

CURRICULUM VITAE………………………………………………………………..130

Page 5: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

v

LIST OF ABBREVIATIONS

5HT 5 Hydroxytryptophan

A2A Adenosine receptor 2A

A3 Adenosine Adenosine receptor 3

AC Adenylyl cyclase

AKHR AKH receptor

AM Adrenomedullin

AMP Adenosine monophosphate

ANOVA Analysis of variance statistical models

AOI Area of interest

AstCR2 Drosophila allatostatin C receptor 2

AT1R Angiotensin 1 receptor

B1AR Adrenergic receptor beta 1

B2AR Adrenergic receptor beta 2

BiFC Biomolecular fluorescence complementation

BK2R Bradykinin receptor 2

BLAST Basic local alignment search tool

BN-PAGE Blue native polyacrylamide gel electrophoresis

BRET Bioluminescent resonance energy transfer

C5aR Complement component 5a receptor

cAMP Cyclic adenosine monophosphate

CCR2b Chemokine receptor type 2b

CCR5 Chemokine receptor type 5

Page 6: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

vi

CFP Cyan flourescent protein

cGMP Cyclic guanosine monophosphate

CGRP Calcitonin gene-related peptide

CHO Chinese hamster ovary cells

CLR Calcitonin-like receptor

Co-IP Co-immunoprecipitation

CPS Counts per second

CRD Cysteine-rich domain

CRE cAMP response elements

CREB cAMP response element-binding protein

CRZR Corazonin receptor

CXCR4 C-X-C chemokine receptor type 4

D2R Dopamine receptor D2

DAF Abnormal Dauer formation

DAG Diacylglycerol

DMEM Dulbecco’s modified Eagle medium

EL Extracellular loop

EPAC Exchange protein activated by cyclic-AMP

ER Endoplasmic reticulum

FRET Fluorescent resonance energy transfer

FSHR Follicle stimulating hormone receptor

GABA Gamma aminobutyric acid

GALR1 Galanin receptor

Page 7: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

vii

GPCR G protein-coupled receptor

GDP Guanosine diphosphate

GEF Guanine nucleotide exchange factor

GFP Green fluorescent protein

GIPs GPCR interacting protein

GIRK G protein-gated inwardly rectifying potassium

GMP Guanosine monophosphate

GnRH Gonadotropin releasing hormone

GRKs G protein-coupled receptor kinases

GRP Gastrin-releasing peptide

GTP Guanosine triphosphate

H1R Histamine receptor 1

H2R Histamine receptor 2

HA Hemaglutinin

HEK Human embryonic kidney cells

IP3 Inositol triphosphate

LH Luteinizing hormone

LK Leucokinin

M1R Muscarinic acetylcholine receptor 1

M3R Muscarinic acetylcholine receptor 3

MAPK Mitogen activated protein kinase

mGlu2R Metabotropic glutamate receptor 2

NFkB Nuclear factor kappa-light-chain-enhancer of B cells

Page 8: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

viii

NK1R Neurokinin 1 Receptor

NK2R Neurokinin 2 Receptor

NKA Neurokinin A

NMU Neuromedin U

NPFR Drosophila NPF receptor

NPY Neuropeptide Y

ORF Open reading frame

OX1 Orexin receptor 1

PCR Polymerase chain reaction

PIP2 Phosphatidylinositol 4,5-bisphosphate

PK1R Drosophila pyrokinin receptor 1

PKA Protein kinase A

PKC Protein kinase C

PLC Phospholipase C

ProcR Proctolin receptor

PSD-95 Postsynaptic density protein 95

RAMPs Receptor-activity modifying proteins

RCP Receptor component protein

RGS Regulators of G-protein signaling

SpIDA Spatial intensity distribution analysis

SPRINP Single primer reactions in parallel

SRE-Luc Serum response element

SSTR2 Somatostatin receptor 2

Page 9: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

ix

T1R1 Taste receptor type 1 receptor 1

T1R3 Taste receptor type 1 receptor 3

T2R Taste receptor type 2

TKR86C Tachykinin receptor at 86C

TM Transmembrane domain

TR-FRET Time-resolved fluorescence resonance energy transfer

TRH Thyrotropin-releasing hormone

TSHR Thyroid stimulating hormone receptor

VFT Venus fly trap domain

WGA Wheat germ agglutinin

YFP Yellow fluorescent protein

α2b-AR Alpha-2B adrenergic receptor

β2AR Beta-2 adrenergic receptor

Page 10: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

x

LIST OF TABLES

Table II.1: Comparison of representative extracellular loop 1 sequences across Class A

GPCR subfamilies………………………………………………………………………77

Table III.1: Receptors utilized in FRET dimer screen………………………………...122

Table III.2: List of primers used for directional cloning of receptor cDNA into pcDNA3

CFP or pcDNA3 YFP expression vectors………………………………………………123

Page 11: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

xi

LIST OF FIGURES

FIGURE I.1: Two-state model of GPCR activation……………………………………54

FIGURE I.2: Functional importance of GPCR heterodimerization…………………….55

FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan

residue exhibits high conservation in Class A GPCR receptor subfamilies…………….79

FIGURE II.2: Mutagenesis of conserved tryptophan residue in LKR ECL1 ablates

receptor signaling………………………………………………………………………..80

FIGURE II.3: Leucine substitution for the conserved tryptophan residue in extracellular

loop 1 leads to a loss of function in multiple receptor types…………………………….82

FIGURE II.4: Substitution of the conserved tryptophan residue to leucine ablates

constitutive activity in a constitutively active AKHR mutant…………………………...84

FIGURE II.5: The WxFG motif is critical for proper receptor trafficking..……………85

FIGURE II.6: Putative tertiary structures of wild type LKR and mutant W101L are

superimposed to identify gross changes in receptor topology…………………………...88

FIGURE III.1: Demonstration of acceptor-photobleaching FRET assay……………..116

FIGURE III.2: Verification of experimental system………………………………….117

FIGURE III.3: Multiple Drosophila Class A neuropeptide receptors exhibit FRET

responses consistent with homodimerization…………………………………………...119

FIGURE III.S1: Verification of signaling in fluorophore tagged receptors..................124

Page 12: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

xii

ABSTRACT

G protein coupled receptors (GPCRs) are a superfamily of transmembrane

proteins responsible for transducing extracellular stimuli into intracellular responses.

GPCRs are indispensable to a vast variety of distinct physiologies and behaviors and

represent approximately 50% of all human drug targets. However, considerable debate

exists as to the structural basis for GPCR activation, with a classical monomeric (two

state model) conflicting with a growing number of reports indicating that these receptors

form higher order functional oligomers. These receptor-receptor interactions can impact

receptor trafficking, ligand sensitivity, desensitization, and strength of effector response.

As such, an understanding of GPCR oligomerization is indispensable to our overall

understanding of receptor dynamics. Additionally, the specific molecular events

underlying receptor activation and signaling remain incompletely understood. Since the

initial discovery of the GPCR receptor family, a number of conserved amino acid motifs

have been identified that have been shown to play specific and critical roles in GPCR

activation, intracellular G-protein coupling, and receptor desensitization. Still, many of

these motifs remain incompletely described, with some motifs having only been

evaluated in a small subset of receptors, and experimental evidence suggests that in some

cases, these conserved motifs may have divergent roles in specific receptor subfamilies.

As such, the conservation of these motifs throughout GPCR evolution represents and

interesting and unresolved aspect of GPCR function.

The goal of this research was two-fold. In one study, I utilized a combination of

bioinformatics, site-directed mutagenesis, signaling assays, and fluorescent microscopy

techniques to evaluate the functional role and evolutionary conservation of a specific

Page 13: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

xiii

amino acid motif, the WxFG motif, which is present in approximately 90% of all Class A

receptors. Our investigation showed that, in contrast to previous studies of this motif,

disruption of the WxFG motif results in trafficking defects across a range of GPCRs

representing multiple Class A GPCR subfamilies, regardless of taxa. A second study

evaluated whether Drosophila GPCRs, specifically a subset of neuropeptide receptors,

assembled as higher order structures at the plasma membrane. While there have been

many receptors shown to assemble as dimers or oligomers at the plasma membrane since

the phenomenon was first recognized over two decades ago, the majority of these studies

focused on vertebrate GPCRs, and the question of whether invertebrate GPCRs show

similar phenotypes has been poorly evaluated, and to date, no Drosophila GPCR has

been empirically demonstrated to assemble as a dimer. To gain a deeper understanding of

GPCR molecular assembly, I evaluated multiple Drosophila receptors utilizing FRET

microscopy to determine both the prevalence of GPCR dimerization among Drosophila

neuropeptide receptors, and determine whether dimerization is conserved across taxa in

specific receptor subfamilies. This investigation showed that all Drosophila GPCRs

tested were able to assemble as homodimers when expressed in a heterologous expression

system, suggesting that not only do Drosophila GPCRs likely assemble as higher order

structures at the plasma membrane, but also that the phenomenon of receptor

dimerization is an ancient property of the receptor superfamily that has been conserved

throughout GPCR evolution. Taken together, these investigations further our

understanding of the molecular events underlying GPCR signaling, and suggest that

many aspects of receptor function are not taxa specific, and are likely fundamental

Page 14: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

xiv

features of GPCR function that have been conserved throughout the evolution of this

receptor superfamily.

Page 15: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

1

CHAPTER I: G protein-coupled receptors– A review of structure-function

relationships critical for receptor signaling.

G protein-coupled receptors, or GPCRs, are the largest cell surface receptor

superfamily in humans1. They are characterized by a conserved molecular structure, with

seven transmembrane domains, an extracellular N terminus, and an intracellular C

terminus. Their primary function is to transduce a variety of extracellular stimuli,

including but not limited to light, ions, small molecules, steroids, and peptides, into

appropriate intracellular responses2. These receptors play a critical role in a variety of

physiologies and behaviors including but not limited to vision, gustation, olfaction, stress

response, cellular communication, reproduction, and development. GPCRs are further

classified based on structural and sequence homology into one of six classes: the Class A,

rhodopsin-like receptors, which represent the largest and most diverse class of GPCRs,

the Class B, secretin-like receptors, the Class C, metabotropic glutamate-like receptors,

the Class D, fungal mating type receptors, the Class E, cyclic AMP receptors found in

Dictostelium slime molds, and the Class F, Frizzled/Smoothened receptors3. Given the

extent that GPCRs mediate cellular communication and function across an incredible

range of biological systems, as well as their therapeutic importance, as approximately

50% of all drugs on market target a GPCR, it is unsurprising that they have been a

significant research focus since the first GPCR was molecularly cloned in 19864,5. This

research, along with the sequencing of multiple genomes , has led to number of

individual GPCRs being identified, with the human genome alone encoding

approximately 800 different receptors1. Despite this, the mechanisms associated with

GPCR activation and signaling remain incompletely understood., Accumulating evidence

Page 16: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

2

shows that many GPCRs exhibit the ability to form dimeric or oligomeric structures with

other GPCRs. Oligomeric association results in a variety of impacts on trafficking,

signaling, and overall function. This observation suggests that GPCRs themselves may

represent allosteric regulators of other GPCRs, and that the functional unit for many

GPCRs may be two receptors assembled as a dimer, rather than individual receptor

monomers6. This phenomenon is further complicated by the fact that a single receptor

may form dimers with either other identical receptors (homodimers) or unrelated GPCRs

(heterodimers), complicating issues such as ligand selectivity and intracellular receptor

coupling. This chapter will serve to review the events associated with GPCR signaling

and common intracellular GPCR pathways, as well as specific amino acid motifs that

have been identified as playing critical roles in GPCR activation and signaling. I will also

review allosteric modulation of GPCRs and GPCR dimerization, as well as discuss the

impact of these phenomena on GPCR function. We will begin with an overview of the

molecular events associated with GPCR activity.

Classical Model of GPCR Activation

The receptor superfamily is named for the intracellular machinery they couple to

– a heterotrimeric G-protein consisting of α and βγ subunits. In the inactive receptor state,

the α subunit of the heterotrimeric G-protein is bound to a GDP molecule, and the α and

βγ remain together in complex with one another. Receptor activation, either through

ligand binding to the extracellular surface of the receptor or other noncanonical

mechanisms (e.g., light, mechanical stimuli) induces a conformational change in the

receptor7. This event allows the receptor to function as a guanine-nucleotide exchange

factor (GEF), removing the GDP bound to the α subunit and replacing it with a GTP.

Page 17: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

3

This GTP binding event activates the α subunit, allowing it to dissociate from the GPCR

and βγ subunits and translocate within the cell to elicit a variety of second messenger

responses8. GPCRs are generally characterized by the α subunit they interact with, the

three most common being Gαs, Gαi/o, and Gαq, which activate different intracellular

signaling pathways.

Signaling through Gα subunits – types and cellular functions

Stimulated Gαs subunits increase the activity of adenylate cyclase (AC), a

membrane bound enzyme responsible for the production of cAMP9. This, in turn,

increases the activity of cAMP-dependent protein kinase (PKA), which phosphorylates a

number of different intracellular targets to elicit a variety of cellular responses, one of the

most notable is the activation of CREB, a transcription factor which binds to cyclic AMP

response elements (CRE) to modulate the transcription of various genes. As such, CREB

activity is a key regulator in a variety of physiologies, such as the suppression of the

oncogene c-fos and the maintenance of circadian rhythms through changes in expression

of timeless and period genes10,11. Changes in intracellular cAMP concentrations also act

to modulate the activity of ion channels, leading to changes in membrane potential and

cell excitability. Cyclic AMP levels also contribute to a number of physiological effects,

such as the mobilization of energy stores through the breakdown of glycogen, stress

response, modulation of heart rate, insect diuresis, and the formation and maintenance of

memory12–16. Cyclic AMP levels also modulate the activity of intracellular exchange

proteins, such as exchange protein activated by cAMP (EPAC), which translocates to the

plasma membrane following activation and produces a range of cell specific responses17.

Page 18: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

4

Taken together, the activation of AC by GPCRs has significant impacts on many aspects

of cell physiology and function.

In contrast to the Gαs subunit, another subset of Gα subunits, Gαi/o, act as an

antagonist to AC, inhibiting the production of cAMP and in turn downregulating PKA

and CREB activity18. Downregulation of cyclic AMP is a hallmark of many inhibitory

neurotransmitters, such as GABA19, and as such serves to regulate a variety of

physiologies and behaviors, including organismal stress responses and sleep onset20. The

combination of both Gαi and Gαs coupled GPCRs in a single cell affords the ability to

precisely regulate intracellular cAMP levels and mediate subsequent downstream effects

through the additive effects of these receptor types.

Another major subset of heterotrimeric G-proteins is the Gαq subunit family. Gαq

subunits function by acting on phospholipase C (PLC), a membrane bound enzyme

responsible for the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol

triphosphate (IP3) and diacylglycerol (DAG)21. IP3 acts on the endoplasmic reticulum,

causing it to release calcium into the cytoplasm, increasing the intracellular concentration

of calcium and in turn increasing the activity of protein kinase C (PKC)22. DAG remains

within the plasma membrane but is a direct activator of PKC and also facilitates PKC’s

translocation to the plasma membrane. PKC phosphorylates a variety of intracellular

targets, leading to significant changes in cellular physiology. These events include

activation of MAPK/ERK pathway, which in turn leads to significant changes in gene

expression and regulation of the cell cycle and cellular proliferation23. Additionally,

many cellular secretion events are calcium dependent, and as such the release of

sequestered calcium ions from the endoplasmic reticulum can lead to the secretion of

Page 19: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

5

neuropeptides and other molecules involved in cellular communication24. PKC also

activates the NFkB protein complex, a key regulator of gene expression with myriad cell-

specific effects, including the suppression of anti-apoptotic genes and the activation of

pro-inflammatory genes25.

Other Gα subunits fulfill cell-specific functions. For example, the activation of

Rhodopsin by light activates the α subunit transducin, which is responsible for the

breakdown of cyclic GMP (cGMP) through modulation of phosphodiesterase activity in

photoreceptor-expressing cells 26. This change in cGMP levels is critical for the

processing of visual stimuli. Similarly, stimulation of gustatory GPCRs activate the Gα

subunit gustducin, a transducin homolog, which increases cGMP degradation. This

signaling is interpreted by the brain as specific tastes, and depends upon cell type27.

Gustducin removes the inhibition of cAMP phosphodiesterase, reducing cAMP

concentrations in taste receptor-expressing cells. The modulation of cAMP and cGMP in

these cells is responsible for the processing and interpretation of taste stimuli, a critical

function which allows organisms to discriminate between palatable and potentially

harmful foods27.

Signaling through Gβγ subunits – types and cellular functions

In addition to Gα subunit dependent signaling, the β and γ subunits also contribute

to GPCR mediated intracellular effects following receptor activation. Unlike the Gα

subunit, which translocates to various intracellular targets in the cytoplasm, the β and γ

subunits remained tethered to the plasma membrane in both their inactive and active

states. Additionally, the β and γ subunits do not dissociate from one another, and as such,

the heterodimer of the two subunits represents the functional unit involved in signaling.

Page 20: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

6

Following receptor activation and the dissociation of the activated Gα subunit of the

heterotrimeric G-protein, the βγ subunit acts on different membrane-bound targets to

elicit cell- and receptor- specific responses. Some of these actions involve the modulation

of a family of G-protein-activated inwardly rectifying potassium (K+) channels, or

GIRKs. The βγ subunits bind directly to intracellular residues on GIRKs, as resolved by

FRET analysis, which in turn activates the ion channel28. This regulation in turn impacts

cell excitability and alters the function of neurons and cardiac muscle29. These

interactions are also facilitated by a family of proteins, the regulators of G-protein

signaling (RGS). RGS proteins are GTPase activating proteins which facilitate GTP

hydrolysis in the Gα subunit and are critical for controlling GPCR signal termination.

Many RGS proteins exhibit significant homology to the γ subunit of the βγ complex and

research has shown that RGS proteins can directly interact with the β subunit of the

heterotrimeric G-protein, forming a β-RGS complex, and potentially replacing the γ

subunit30. Additionally, recent work has shown that RGS-insensitive mice display

significantly reduced GIRK activity when the μ-opioid receptor was activated, further

indicating a functional association of these proteins and inhibition of βγ dependent

signaling31. βγ subunits inhibit the actions of multiple voltage-gated calcium (Ca2+)

channels in a voltage independent manner28. As these channels are primarily expressed in

neurons, this inhibition can prevent the occurrence of action potentials in these cells, and

in turn modulate the activity of various neural circuits and their corresponding behavioral

and physiological outputs.

βγ subunits have also been shown to directly interact with second-messenger

producing molecules such as PLC and adenylate cyclase (AC) to modulate second

Page 21: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

7

messenger production in a similar manner to Gα subunits. βγ subunits act directly on AC

to activate or inhibit the production of intracellular cAMP both in concert with or

opposing the actions of corresponding Gα subunits32, which can in turn modulate cAMP

dependent signaling pathways described previously such as CREB activated gene

transcription. In a similar manner, βγ subunits also bind directly to phospholipase C

isoforms to both activate and inhibit PLC activity 33,34. This in turn modulates IP3 and

DAG production, and subsequent calcium release from intracellular endoplasmic

reticulum stores. Taken together, βγ subunits play a major role in regulating cell-

excitability and physiological changes stemming from GPCR activation.

GPCR signal termination and endosomal signaling

GPCR signal termination involves both the sequestration of the receptor-ligand

complex from the cell surface and the termination of G-protein signaling. G-protein

signaling is terminated through the innate GTPase activity which Gα subunits possess35.

As such, the bound GTP molecule is hydrolyzed following activation and the molecule

loses its enzymatic activity and returns to an inactive state upon GTP hydrolysis. This

facilitates the re-association of the Gα and Gβγ subunits of the heterotrimeric G protein

complex. Next, sequestration of the activated G-protein coupled receptor is facilitated by

phosphorylation events following the dissociation of the heterotrimeric G-protein

complex and the recruitment of arrestins, a family of cytoplasmic proteins responsible for

targeting GPCRs to early endosomes35. Specifically, following the dissociation of the

heterotrimeric G-protein complex, phosphorylation sites on the intracellular face of the

GPCR are exposed. These sites are phosphorylated by a family of serine/threonine

kinases known as G-protein coupled receptor kinases (GRKs). Phosphorylation of the

Page 22: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

8

receptor causes the recruitment of arrestins to the GPCR. Arrestins act to block GPCR

signal transduction in two major ways: first, by physically obstructing the association of

heterotrimeric G-proteins with the activated GPCRs, and second, targeting the arrestin-

GPCR complex to clathrin-coated pits for eventual removal of the GPCR from the

plasma membrane35. The GPCR is encapsulated into an endosome and the fate of the

GPCR is either to be recycled back to the plasma membrane following receptor-ligand

dissociation, or ultimately targeted for degradation via lysosomes. These internalization

events alter the overall sensitivity of the cell to the specific GPCR ligand by decreasing

the number of receptor molecules present at the plasma membrane, a process known as

desensitization, although it should be noted that visual rhodopsin undergoes

desensitization through a different mechanism36. Additionally, recent evidence suggests

that some GPCRs signal directly through the arrestin-endosome complex, indicating that

arrestins may serve in both GPCR signal termination as well as signal transduction, with

specific roles including the activation of the MAPK/ERK pathway, and the inhibition of

nF-kB activity37. Specifically, the β-arrestin 1 subunit regulates the thyrotropin-

stimulating hormone receptor’s (TSHR) downstream effects on cholesterol metabolism38.

Additionally, recruitment of arrestin to the D2 dopamine receptor (D2R) is responsible for

cocaine-induced hyperlocomotion, but not incentive motivation in this experimental

paradigm39. Taken together, this suggests that the arrestin-endosome complex may

function along with canonical cAMP/Ca2+ pathways to mediate the full array of GPCR

dependent intracellular signaling.

Page 23: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

9

The “two-state” model and its shortcomings

The predominant mechanism used to describe GPCR activation is the “two-state

model”, wherein the receptor occupies two distinct states, R(inactive) and R*(active)

(Figure 1). In this model, the inactive receptor exists at the plasma membrane coupled to

intracellular heterotrimeric G-proteins. Ligand binding to the extracellular face of the

receptor induces conformational changes throughout the receptor, allowing it to act as a

guanine-nucleotide exchange factor (GEF) and in turn facilitate the “swapping” of a

bound GDP molecule for a GTP molecule from the alpha subunit of the heterotrimeric G

protein associated with the receptor. This event activates the heterotrimeric G-protein

complex, allowing it to dissociate from the receptor and act on myriad intracellular

targets to transduce the extracellular signal generated by the ligand into an appropriate

cellular response. Signal termination results from the innate GTPase activity of the Gα

subunit, as it eventually hydrolyzes GTP to GDP and returns to an inactive state. While

this model accounts for the basics of GPCR activation and signal transduction, it has

become clear that the two-state model first proposed for GPCR activation fails to account

for a great deal of reported results since its first proposal8.

GPCRs exhibit a classic sigmoidal dose-response curve for both ligand binding

and receptor activity, which suggests allosteric modulation of receptor signaling and

cooperativity. If, in fact, GPCRs only occupied two conformational states – ligand-bound

and ligand-unbound, dose response curves for ligand binding and receptor activity should

be hyperbolic, rather than sigmoidal. Indeed, many allosteric modulators of GPCR

signaling have been identified in the past three decades, collectively referred to as GPCR

interacting proteins (GIPs) that in many cases are critical for proper receptor function40.

Page 24: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

10

For example, the human calcitonin-like receptor (CLR) requires the interactions of

multiple GIPs to appropriately transduce signals from its endogenous ligands

adrenomedullin (AM) and calcitonin gene-related peptide (CGRP)41. First, the receptor

must associate with receptor activity modulating proteins (RAMPs) to efficiently traffic

to the plasma membrane. Coupling to RAMP1 confers greater receptor specificity to

CGRP, while coupling to RAMP2 or RAMP3 confers greater receptor specificity to AM,

suggesting that these allosteric interactions are capable of modifying the CLR ligand-

binding site to alter specificity to these disparate ligands42. Additionally, CLR must

interact with a second allosteric modulator, receptor component protein (RCP), to

transduce signals from either ligand. Thus, the receptor not only requires allosteric

interactions for proper signal transduction, but is also able to change its conformational

state to selectively bind one ligand over another, and must therefore exhibit multiple

active states.

Another shortcoming of the two-state model is its presupposition that, in a ligand-

unbound state, the receptor exists in an “off” conformation incapable of activating an

associated heterotrimeric G-protein. In reality, many GPCRs exhibit constitutive activity

even in the absence of ligand, suggesting that many GPCRs do not exist in a truly “off”

state when expressed43,44. This has led to speculation that GPCRs act as “rheostats” rather

than simple on-off switches45. Under this paradigm, GPCRs can exist in multiple

conformational states with multiple intermediate states between “inactive” and “active”.

The exact state then would result from intramolecular interactions within the receptor,

allosteric interactions with other proteins, ligand binding, and ligand identity45. Some

GPCRs have also been shown to activate multiple intracellular G protein pathways, an

Page 25: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

11

example of this being the gonadotropin releasing hormone (GnRH) receptor, which

potentiates both cyclic AMP and Ca2+ signaling when activated, suggesting that the

receptor is capable of adopting multiple conformations to specifically accommodate

multiple G proteins and intracellular signaling pathways46. This selectivity in G protein

recruitment can also be controlled by ligand identity, as is the case with the neurokinin 2

(NK2) receptor. NK2 endogenously binds to neurokinin A (NKA), a gene product of the

preprotachykinin gene that also gives rise to substance P47. Full length NKA elicits both

calcium and cAMP responses from NK2 receptor activation, yet a C-terminally truncated

form of NKA (NKA4-10) only elicits calcium responses upon binding to NK2,

suggesting that this receptor is also capable of adopting multiple active conformations in

a ligand-dependent manner48. Indeed, the idea of a GPCR occupying multiple “activated”

conformations has gained considerable traction and is supported by structural modeling,

however no GPCR crystal structures for a single receptor bound independently to

multiple ligands have yet been determined49.

Conserved Amino Acid Motifs in Class A GPCRs

As our understanding of GPCRs has grown, a number of conserved amino acid

motifs have been identified which are critical for discrete aspects of receptor function.

Amino acid motifs are sequences of amino acids that exhibit both high sequence

conservation within a specific protein family, and generally participate in a common

function. For GPCRs, the majority of these motifs have been identified and described for

Class A GPCRs, which is not unsurprising given that Class A GPCRs are the largest

GPCR subclass. For Class A GPCRs, conserved amino acid motifs identified and

described to date include the E/DRY motif located at the base of TM3, the WxFG motif

Page 26: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

12

located in EL1, the CWxP motif located in TM6, and the NPxxY motif located on TM7.

Much of our understanding of the function of these motifs is derived from mutagenesis

studies, wherein conserved amino acid residues are mutated and resulting changes in

receptor function are documented. These approaches, coupled with GPCR crystal

structure analysis, have led to each of these conserved amino acid motifs being associated

with specific aspects of receptor function. In this section, I will briefly review common

aspects of receptor function associated with these specific motifs.

E/DRY motif – The Ionic lock

The E/DRY motif, located at the base of TM3, was first identified in Rhodopsin

and later functionally characterized through investigations of the β2 adrenergic receptor

(β2AR)50. The most critical role for this motif is stabilizing the inactive conformation of

Class A receptors through interactions between the positively charged arginine residue

(conserved in 96% of Class A GPCRs) in the DRY motif in TM3 and a conserved,

negatively charged amino acid (usually glutamate or aspartate) in TM651. This interaction

forms an “ionic lock” which keeps TM3 and TM6 in close proximity to one another when

the receptor is not bound to ligand. Ligand binding is thought to break this lock through

conformational changes in the receptor, leading to TM6 moving away from TM3 and

forming an intracellular pocket through which the GPCR can interact with and activate

heterotrimeric G proteins52. This hypothesis is supported by multiple lines of evidence.

First, increased distance between TM3 and TM6 is correlated with higher constitutive

receptor activity in β adrenergic receptors53. Additionally, multiple studies have shown

that disruption of the ionic lock through mutagenesis leads to an increase in constitutive

activity, lending further support to the notion that this motif serves to stabilize the

Page 27: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

13

inactive receptor state54–56. A salt bridge formed between the aspartate and arginine

residues in the DRY motif has been identified in crystal structures of inactive GPCRs,

and disruption of this interaction is a critical event related to receptor activation,

implicating this motif in multiple receptor-stabilizing interactions57. In addition to its

apparent role in stabilizing the inactive receptor state, it has been reported that the

conserved arginine in the DRY motif is responsible for coupling the receptor to

intracellular G-proteins, with a naturally occurring mutation (Arg � His) in the human

vasopressin type two receptor giving rise to a receptor incapable of stimulating adenylate

cyclase, resulting in persistent nephrogenic diabetes insipidus in individuals bearing this

mutation58. A similar Arg � His mutation in the human gonadotropin releasing hormone

(GnRH) receptor leads to hypogonadotropic hypogonadism, but in contrast to the

vasopressin receptor, this mutant receptor is unable to bind ligand, suggesting that this

motif may exhibit receptor specific functions beyond stabilization of the inactive receptor

state59.

WxFG motif – an extracellular domain critical to receptor trafficking

Another amino acid motif that has been shown to play a critical role in overall

receptor function is the WxFG motif found on extracellular loop 1 (EL1)60. This motif,

initially described by Klco et al. in 2006, is relatively understudied in comparison to the

extensive literature exploring the DRY motif, but appears to play a critical role in proper

trafficking of the receptor from the ER-Golgi complex to the plasma membrane60,61.

Present in approximately 90% of Class A GPCRs, the WxFG motif was originally

reported to play a critical role in ligand-mediated activation of the C5a complement

component receptor. Mutagenesis of the conserved tryptophan residue in this domain

Page 28: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

14

yielded a nonfunctional C5a receptor unable to respond to ligand, but apparently did not

impact ligand binding, as purified membranes containing W102A mutant C5a receptor

variants were still capable of binding ligand (although only at ~20% of wild type receptor

maximal occupancy), but did not transduce ligand binding into an appropriate

intracellular response61. A W102F C5a receptor variant was capable of binding both

ligand and activating intracellular responses at levels nearly indistinguishable from wild

type, prompting the hypothesis that an aromatic, bulky amino acid at the “W” position

was necessary for wild type receptor function61.

A subsequent study by our laboratory of eight Class A GPCRs from disparate

subfamilies by our laboratory instead showed that this motif was critical for wild type

receptor trafficking60. Using a comparison of wild type and WxxxL mutant receptors

fused to a C-terminal YFP molecule, we showed that not only were WxxxL mutants

incapable of responding to ligand, but that these receptors remained trapped in the ER-

Golgi complex and were not appropriately trafficked to the cell surface if an aromatic

amino acid was not present at the “W” position60. These findings, while seemingly in

conflict with the original report on C5aR receptor function, can be reconciled through a

comparison of the methodologies used in these two studies. While Klco et al. showed that

the W102A mutant C5a receptor did not signal in response to ligand, ligand binding to

the receptor was performed on purified cell membranes, which would have captured

receptors trapped in the ER-Golgi complex as well as the plasma membrane. Thus, the

ability of the receptor to still bind ligand is largely unimportant as it appears mutant

receptors do not reach their appropriate intracellular target (the plasma membrane) in

order to respond to extracellular ligand presentation. A possible mechanism for this

Page 29: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

15

trafficking defect was suggested by our research, as computer modeling of wild type and

WxxxL mutant receptors showed distortion of the receptor’s natural conformation, with

the N terminus apparently “pushed” further away from the body of the receptor. Other

reports suggest that this particular conserved tryptophan residue in EL1 interacts with and

potentially stabilizes a cysteine mediated disulfide bridge between EL1 and EL2 in

crystal structures of the β2AR, and thus receptor mutants may exhibit overall instability

compared to wild type62. It is also important to note that a conserved cysteine residue in

EL1 downstream from the WxFG motif has been previously shown to form a disulfide

bond with another conserved cysteine residue helps maintain receptor architecture, and it

could be the case that mutagenesis of the WxFG motif interferes with this disulfide bond

formation63. Taken together, these reports suggest that this motif plays a critical role in

GPCR function through appropriate trafficking of the receptor to the plasma membrane,

while further research is necessary to fully determine the molecular mechanisms involved

with this process.

CWxP motif – “rotamer toggle switch”

A third amino acid motif critical to GPCR function is the CWxP motif, present on

TM6. This motif is highly conserved amongst class A GPCRs, with cysteine and

tryptophan conserved in over 70% of Class A, non-olfactory GPCRs, while proline is

conserved in a remarkable 98% of non-olfactory receptors64. Crystal structure and

molecular modeling studies have shown that this conserved proline residue induces a

large bend in TM6, whose outward motion during receptor activation contributes to the

formation of the G-protein binding pocket on the intracellular face of the receptor65,66.

Mutagenesis studies on the β2AR revealed that this outward motion away from TM3 is

Page 30: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

16

also associated with a change in the rotamer state of the conserved cysteine and

tryptophan residues in this motif, giving rise to its classification as a “rotamer toggle

switch”66. This change in side chain orientation is hypothesized to stabilize the receptor

in an active conformation. The functional role of this motif in receptor activation is

further supported by studies of the thyrotropin stimulating hormone receptor, where

substitution of arginine for the conserved cysteine residue in this motif was associated

with increased constitutive receptor activity67.

Still, the function of this motif does not appear to be universal amongst class A

GPCRs. While crystal structure studies of the β2AR indicate that the conserved

tryptophan residue in this motif does adopt different side chain orientations in active

receptor conformations compared to inactive states, the crystal structure of a

constitutively active rhodopsin receptor did not find a similar correlation with the rotamer

position of the conserved tryptophan residue and active or inactive receptor

conformations68,69. This, along with the absence of this conserved tryptophan residue in

~30% of Class A GPCRs, suggests that this rotamer switch model of receptor activation

may not be uniform amongst Class A GPCRs, and other mechanisms must therefore

stabilize the transition between active and inactive receptor states. The dearth of

available GPCR crystal structures of receptors in an active conformation further

complicates this, as it is currently impossible to determine the prevalence of rotamer

rearrangements of CWxP motif residues amongst all class A GPCRs. Still, the high

degree of conservation exhibited by this motif and experimental evidence from β2AR

investigations suggest that it does play a critical role in GPCR function in at least a subset

of Class A GPCRs.

Page 31: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

17

NPXXY – a conserved motif with multiple functional roles

Another conserved amino acid motif that has been shown to play a significant role

in GPCR function is the NPXXY motif found on the base of TM7. This motif exhibits

remarkable conservation amongst GPCRs, with the conserved tyrosine residue being

present in 92% of all class A receptors70. Early studies on the β2AR identified that the

conserved tyrosine in this motif was necessary for agonist-induced receptor

desensitization, with Tyr�Ala mutant receptors exhibiting no internalization in response

to prolonged agonist exposure71. This mutant receptor exhibited no significant defects in

ligand binding or adenylate cyclase activation, suggesting a singular role for this motif in

arrestin-mediated desensitization. Interestingly, mutations of the conserved asparagine

and proline residues to alanine in the same β2AR resulted in a significant reduction in

ligand sensitivity, suggesting that this motif may stabilize the inactive receptor state to

facilitate ligand binding in addition to its role in receptor desensitization72. However,

further studies of disparate GPCRs indicated that this motif did not play a universal role

in GPCR desensitization. Substitutions of the conserved tyrosine residue in the B2

Bradykinin receptor resulted in constitutive internalization of the receptor, along with a

loss of signaling capabilities73. Additionally, the gastrin-releasing peptide (GRP) receptor

showed no significant defects in receptor internalization following a similar Tyr�Ala

substitution, indicating that the conserved tyrosine residue in this motif is unlikely to

mediate receptor desensitization in all class A GPCRs74.

More recent reports suggest that, similar to the CWXP motif, the conserved

tyrosine residue in this motif may function as a “toggle switch” contributing to the

receptor adopting an active conformation following ligand binding70. Specifically, crystal

Page 32: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

18

structure studies of opsin molecules thought to mimic the active state of rhodopsin have

indicated that, in the active state of the receptor, the conserved tyrosine residue changes

its rotamer configuration and inserts into the space occupied by TM6 in the inactive

receptor conformation. This conformational change is believed to stabilize the active

conformation of the receptor75. This hypothesis is further supported by a recent

investigation of the α1b and β2 adrenergic receptors that demonstrated reduced signaling

through targeted mutagenesis, and is consistent with a stabilization of the inactive state of

these receptors76. It is important to note that previous studies of the β2AR showed that

mutagenesis of this conserved tyrosine residue to alanine resulted in desensitization

defects, suggesting that amino acid identity at this position plays a critical role in wild

type receptor function. Given the distinct functional roles of this motif in different

receptor backgrounds, further investigation is necessary to determine whether the

NPXXY motif exhibits a conserved functional role in all class A GPCRs, or whether its

function is receptor-specific.

GPCR dimerization – allosteric modulation of GPCR signaling through receptor-

receptor interactions.

Efforts to fully elucidate GPCR function are complicated by the property of many

receptors in this family to form higher order structures: dimers and oligomers, with other

GPCR members. Since the first GPCR dimer was reported in 1998, multiple studies

describe that many GPCRs exhibit extensive dimerization, at times with multiple GPCR

subtypes, and these events lead to alteration in signaling, ligand selectivity, receptor

desensitization, and other aspects of receptor physiology. This section will review GPCR

Page 33: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

19

oligomerization and discuss the functional consequences of these interactions in overall

GPCR function.

The earliest functional characterization of GPCR dimerization was described in

GABAB receptors. Specifically, the GABABR1 gene product, when heterologously

expressed in HEK293T cells, remained trapped in the endoplasmic reticulum. However,

when GABABR1 was coexpressed with the GABABR2 receptor encoded by a different

gene, GABABR1 cell surface expression was observed77. Subsequent work. revealed the

mechanism for this phenomenon – dimerization between GABABR1 and GABABR2

masked a C-terminal RXR ER retention motif present on the GABABR1 receptor78. Thus,

the functional receptor for the GABAB receptor subclass was revealed to be a heterodimer

of two related receptors, rather than a receptor protomer, providing the first evidence for

a functional role of dimerization in GPCR signaling.

Since these initial studies, a wealth of data has supported the capacity of multiple

GPCRs to assemble as dimers and higher order oligomers. This section will expound on

the mechanisms of dimerization and the functional implications of this phenomenon.

Mechanisms of GPCR dimerization

The molecular mechanisms underlying GPCR dimerization differ by receptor

class, the most well characterized of which is the Class C, metabotropic glutamate-like

receptors. These receptors contain an extended N-terminal domain known as a Venus

flytrap (VFT) module, which is unique amongst GPCR subclasses and serves a number of

different functions for Class C receptors. In contrast to Class A receptors, where TM

domains contribute to ligand recognition, Class C GPCRs ligand binding occurs solely

Page 34: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

20

through this extended N-terminal VFT domain79. Interestingly, this same VFT domain is

responsible for forming the dimerization interface for most Class C receptors, which

function as obligate dimers 80. Most Class C GPCR N-termini contain multiple cysteine-

rich domains (CRDs), located between the VFT and TM1, which are able to crosslink the

receptor to its dimeric partner through interactions between these extended N-termini,

leading to the formation of stable receptor dimers81. Exceptions to this disulfide-linkage

mechanism do exist, most notably exemplified by the GABAB receptors. The N-termini

of these receptors, while involved in ligand binding similar to other Class C receptors,

lack CRDs. As a result, dimerization for these receptors cannot involve N-terminal

disulfide linkages, rather, dimerization occurs through interactions between the C-

terminal tails of GABAB receptors. The RXR ER-retention motif found on GABABR1

binds to an unrelated coiled-coil domain found on the C-terminus of GABABR2, masking

this domain and allowing for the functional expression of the GABAB heterodimer at the

cell surface81.

In contrast to the rather uniform mechanisms underlying Class C GPCR

dimerization, our understanding of the interfaces underlying Class A and B receptor

dimerization remains incomplete. Dimerization between receptors of these two classes

does not involve covalent linkages between dimer partners, as is often found in Class C

receptors, and as such, these receptors often exist as transient, rather than stable, dimers

at the cell surface82. Dimerization of Class A GPCRs often relies on interactions between

TM domains of the receptors involved, with multiple interactions between disparate TM

regions having been reported. Crystal structure analysis of the chemokine receptor

CXCR4 showed that the receptor assembles as homodimer with an interface involving

Page 35: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

21

both TM5 and TM6 from each receptor subunit83. Here, dimer stabilization is mediated

primarily through hydrophobic interactions between residues in these domains83. In

contrast, disulfide-trapping experiments on the unrelated 5HT2c receptor also showed a

homodimeric interface involving TM5 of each protomer, but rather than TM6 also

contributing to dimerization as seen with the CXCR4 receptor, the 5HT2c homodimer

involves interactions between TM4 domains in each dimeric counterpart, constituting an

overall TM4/TM5 dimeric interface between the receptors84. Recent work on the

adenosine A2A receptor and D2 dopamine receptor heterodimer suggests that a similar

interaction between TM4 and TM5 on these receptors stabilizes the heterodimer85.

Additionally, atomic force microscopy investigations of mouse rhodopsin also suggested

that the receptor dimerized through TM4-TM5 interactions between receptor protomers86.

Interestingly, crystal structure analysis of the human Class F smoothened receptor also

showed that the receptor formed a homodimer similarly stabilized by interactions

between TM4 and TM5, indicating that this interface may be common to multiple GPCR

homo- and heterodimers, regardless of class87. Such an interaction likely arose early in

GPCR evolution, given the significant evolutionary distance between Class A and Class

F GPCRs88. Beyond the TM5-TM6 and TM4-TM5 interfaces described above, additional

Class A GPCR dimeric interfaces have been identified. Crystal structure analysis of the

B1 adrenergic receptor (B1AR) revealed a TM1-TM2-C terminus interface responsible

for stabilizing the B1AR homodimer, along with the previously described TM4-TM5

interface89. Taken together, these findings suggest that Class A GPCRs dimerize through

a variety of dimerization interfaces, with the specific receptor regions involved likely

varying greatly amongst the superfamily.

Page 36: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

22

In contrast to Class A and Class C GPCRs, limited evidence exists regarding

mechanisms underlying Class B GPCR dimerization. A study utilizing spatial intensity

distribution analysis (SpIDA) on the human secretin receptor revealed a critical role for

TM4-TM4 interactions in stabilizing secretin receptor dimers, with mutagenesis of amino

acid residues within this domain significantly reducing receptor dimerization90.

Interestingly, a similar role for TM4-TM4 interactions in stabilizing Class B receptor

dimerization was found in an investigation of rabbit calcitonin receptor homodimers, a

related Class B receptor91. These findings suggest that a common TM4-TM4 dimeric

interface may unify Class B GPCR dimerization, though this hypothesis requires

evaluation in a greater diversity of Class B receptors before it can be fully supported. It is

interesting to note that, for each receptor subclass, disparate receptor domains seem to

contribute to higher order assembly, and as such, it is unlikely a unifying mechanism of

dimerization exists for GPCRs. Additionally, in contrast to Class A, B, and C GPCRs,

putative dimerization domains for Class D, E, and F GPCRs, beyond the smoothened

receptor, have not been explored, and as such, models for dimerization amongst these

receptor types remains incomplete.

Impacts of GPCR dimerization on receptor function

Many reports have shown that GPCR dimerization impacts receptor function and

signaling, highlighting the necessity of identifying homo- and heterodimer GPCR

complexes. The nature in which oligomerization impacts function exhibits incredible

diversity, particularly amongst heterodimeric complexes, which will be discussed herein.

Page 37: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

23

Allosteric modulation through homodimerization

Many of the effects associated with GPCR dimerization, including receptor trans-

activation and trans-desensitization, are absent in GPCR homodimers as a result of

identical protomers constituting the dimeric unit. Still, evidence suggests that

homodimeric GPCRs do not signal similarly to monomeric counterparts, and

homodimeric assembly impacts overall receptor function. The strongest evidence for this

supports homodimeric GPCRs functioning as negative allosteric modulators to their

protomeric counterpart. In this model, ligand binding to one of the two protomers in a

GPCR homodimer decreases the likelihood of a second ligand binding. This phenomenon

has been shown in multiple GPCRs, including the human thyrotropin (TSH) and

luteinizing hormone (LH) receptors, as well as the A3 adenosine receptor92,93. This

phenomenon of negative cooperativity among dimer partners is not limited to

homodimers, and has been observed in chemokine receptor heterodimers involving the

CCR5 and CCR2b receptors, suggesting that allosteric modulation of dimer partners is a

hallmark of GPCR dimerization in general94. These findings are interesting as they

suggest that dimerization may explain, at least in part, the sigmoidal dose-response

curves associated with GPCR ligand binding and signaling. Negative cooperativity

among dimer partners has been demonstrated in native tissues, in addition to the cell

culture systems commonly used. A study of oxytocin receptors in rat mammary glands

utilizing time-resolved (TR) FRET indicated that not only did these receptors assemble as

homodimers in vivo, but additionally that agonist-binding to a homodimer decreased

overall receptor affinity for agonist, suggesting that, once a receptor dimer has bound a

ligand molecule, its affinity for binding an additional ligand molecule is significantly

Page 38: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

24

decreased95. This phenomenon of negative cooperativity has interesting implications for

GPCR signaling and function. Negative cooperativity amongst GPCR homodimers could

serve to buffer cells against abrupt increases in extracellular ligand concentration96. This

would effectively alter a cell’s affinity for ligand in a concentration-dependent manner,

preventing overstimulation in response to excessive ligand presentation, although this has

never been empirically demonstrated. It is interesting to note that, while theoretically

possible, no instance of positive cooperativity, which could potentially intensify cellular

responses to extracellular ligands, has been demonstrated for any GPCR dimers to date.

Trafficking

GPCR dimerization has also been shown to play a significant role in receptor

trafficking and cell-surface targeting in multiple receptors. The best example of this

phenomenon is the previously mentioned GABAB receptor heterodimer, wherein

dimerization is required for cell-surface expression of the mature receptor77,78. The

necessity of receptor dimerization for wild-type receptor function has since been

demonstrated in multiple additional Class C GPCRs, including the T1R taste receptor and

metabotropic glutamate 2 (mGlu2) receptors, suggesting that this is a hallmark of the

receptor subfamily97–99. In contrast, numerous studies of Class A and B GPCRs, when

forcibly expressed as monomers in detergent micelles or reconstituted nanodiscs, are still

capable of both ligand binding and receptor activation, suggesting that dimerization as a

requirement for receptor trafficking and function may be limited to Class C

receptors97,100. This is likely explained by the mechanical differences that distinguish

Class C receptor dimer formation from other GPCR subclasses. Class C receptor dimers

are stabilized by disulfide bonds between individual protomer molecules, which are

Page 39: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

25

formed during receptor maturation in the ER101. In contrast, Class A receptors, largely

stabilized by hydrophobic interactions between residues found in TM regions of the

receptor, likely exist as a dynamic population of monomers, dimers, and higher order

oligomers at the cell surface, with relatively short half-lives in each structure, and as such

it seems less likely that receptors would co-traffic as dimers during maturation102.

Interestingly, dimerization of the Class A 5HT2c receptor was observed in both the ER

and Golgi complexes during receptor maturation when expressed in HEK-293 cells,

suggesting a possible role for receptor dimerization in trafficking of Class A receptors,

but additional study is necessary to determine whether this is required for cell-surface

expression of a functional receptor103. Still, the available evidence suggests that the

necessity of GPCR dimerization for proper GPCR trafficking is limited to Class C

receptors.

Receptor transactivation

A particularly interesting phenomenon related to GPCR heterodimers is receptor

transactivation. In this scenario, ligand binding to one protomer in a GPCR heterodimer

can lead to signaling through the other protomer (Figure 2). One exemplar of this

phenomenon is the human bradykinin receptor 2 (BK2R) – B2AR heterodimer. When

expressed independently, BK2R signals through the Gαq intracellular pathway, while

B2AR acts through the Gαs signaling pathway104,105. However, when these receptors are

coexpressed, bradykinin stimulation results in signaling through both Gαq and Gαs

intracellular pathways in a B2AR dependent manner, suggesting that Gαs stimulation

results from transactivation of the B2AR protomer106. Interestingly, isoproterenol

stimulation of cells co-expressing these two receptors did not result in activation of the

Page 40: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

26

Gαq, suggesting that transactivation of this receptor pair exhibits asymmetry. The

phenomenon of transactivation is also demonstrated in the GABABR1/GABABR2

receptor heterodimer, where ligand binding to GABABR1 stimulates intracellular

signaling through its partner, GABABR277. Similar transactivation was identified in an

investigation of the luteinizing hormone receptor (LHR). Specifically, ligand-binding

deficient and signaling deficient variants of LHR were co-expressed in populations of

HEK-293 cells. When these variants were co-expressed, wild type luteinizing hormone

signaling was restored, whereas no LH signaling was observed in cell populations

expressing a single mutant receptor variant107. These findings suggest that the LHR

homodimer exhibits transactivation following ligand binding to a single protomer. The

possibility of transactivation between individual protomers of a GPCR heterodimer

potentially complicate assigning a canonical signaling pathway to an individual GPCR, as

non-canonical signaling through transactivation could result in cell- and tissue- specific

responses to particular ligands.

Transdesensitization

In addition to transactivation, GPCR dimerization has also been implicated in

receptor transdesensitization, wherein ligand binding to one receptor in a heterodimer

facilitates the internalization of its heterodimeric partner (Figure 2). This phenomenon is

best exemplified by the μ-opioid receptor/CCR5 chemokine receptor heterodimer pair.

When these receptors are co-expressed in CHO cells, bidirectional transdesensitization

was observed, with μ-opioid receptor pre-stimulation ablating CCR5-dependent

chemotaxis responses, and similar μ-opioid receptor mediated chemotaxis ablated in cells

pre-treated with CCR5 agonists. Interestingly, receptor desensitization was not mediated

Page 41: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

27

through receptor internalization, as CCR5 ligand stimulation did not significantly impact

μ-opioid receptor internalization and vice versa. The phenomenon of trans-desensitization

is also exemplified by the H1 and H2 histamine receptor heterodimer. Upon expression of

these two receptors in CHO cells, pre-incubation with the H1R ligand 2,3-

trifluoromethylphenylhistamine abolished the subsequent H2R responses to amthamine,

an H2R agonist108. Similar to the phenomenon of trans-activation, trans-desensitization

provides non-canonical regulatory mechanisms for the cell to fine tune cellular responses

to extracellular signals.

Ligand sensitivity and biased signaling

In addition to intracellular signaling, dimerization can also differentially impact

ligand selectivity. In mammals, specific combinations of GPCRs homo- or heterodimers

are responsible for detecting bitter, umami, and sweet taste sensations 98. Homodimers of

one of the two main families of GPCR gustatory receptors, Tas2Rs or T2Rs, are

responsible for transducing bitter taste sensation. In the other family of mammalian

gustatory GPCRs heterodimers formed between T1R family members are responsible for

transducing sweet (T1R2-T1R3 heterodimer) and umami (T1R1-T1R3) tastes. This example

indicates how dimerization amongst these receptors can play a critical role in determining

ligand sensitivity. GPCR dimerization has also been shown to impact intracellular

signaling. In the case of the Ciona intestinalis GnRH receptors, heterodimerization

between GnRH receptors significantly alters second messenger production following

ligand challenge109.

Heterodimerization of GPCRs has also been implicated in neuronal signaling and

mood disorders, particularly schizophrenia, as in the case of the 5HT2AR-mGlu2R

Page 42: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

28

heterodimer110. Researchers found that disruption of the 5HT2AR-mGlu2R heterodimer

through knocking out the mGlu2R receptor in mice led to a loss of 5-HT induced

behaviors when challenged with hallucinogenic 5-HT receptor agonists111. This particular

heterodimer is significantly upregulated in post-mortem brain tissue from schizophrenic

patients when compared to normal brains, further suggesting a critical physiological role

for this heterodimer in mood management and sensory perception110.

Methods to detect GPCR dimers and oligomers

A multitude of experimental approaches have been developed or adapted to detect

GPCR dimerization in living cells. These methods include biochemical approaches, such

as co-immunoprecipitation (Co-IP), and microscopy analysis, such as Fluorescent

Resonance Energy Transfer (FRET), Bioluminescent Resonance Energy Transfer

(BRET), and Biomolecular Fluorescent Complementation (BiFC). In most cases, a

combination of methodologies is employed to diminish the likelihood of false positive

reports of dimerization amongst GPCRs. These approaches, and their respective benefits

and pitfalls, will be explored in the following section.

Biochemical resolution of GPCR dimers.

Since their initial discovery, the most common biochemical approach to detect

GPCR dimers and higher order oligomers has been co-immunoprecipitation analysis112.

Co-immunoprecipitation involves epitope tagging one or both GPCRs suspected of

forming a dimer, with the most commonly utilized epitopes for this approach being HA,

FLAG, and Myc. Receptors are then expressed in heterologous cell systems or transgenic

organisms. Tissue is then harvested and placed in a column containing an antibody

Page 43: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

29

against one of the epitope tags found on the modified receptors. Following pull-down, the

bound protein fraction is then eluted and resolved through Western blot analysis, where

the second receptor in the suspected dimer is probed with an epitope or receptor-specific

antibody. Positive results indicate association between the two receptors within the cell.

There are multiple potential pitfalls regarding a Co-IP approach to resolve GPCR

dimers. A positive result is not a definitive indication that the receptors

involved directly interact with one another – the possibility exists that these receptors are

integrated in a larger complex by other proteins. Additionally, GPCRs are transmembrane

receptors, and are notoriously difficult to work with through Western blot analysis. Also,

receptor expression in native tissue may be too low to resolve through Co-IP and Western

Blot analysis, and as such, this approach is best suited to cell-culture systems where

receptor overexpression may occur, thus opening the possibility that assays may capture

interactions that do not occur when receptors are expressed at physiological levels in

native tissues. As such, Co-IP approaches are often complemented by other

methodologies, such as FRET, to increase confidence in findings from such studies.

In addition to co-immunoprecipitation, a less common biochemical approach used

to resolve GPCR dimers is blue native polyacrylamide gel electrophoresis (BN-PAGE).

This methodology utilizes weak detergents, unlike SDS commonly utilized in

conventional Western blot analysis, to preserve protein complexes in their native state,

eliminating the need for a co-immunoprecipitation column113. Coomassie blue dye is also

added to samples in BN-PAGE to confer a negative charge to proteins, as this dye does

not disrupt multiprotein complexes, allowing for their resolution through gel

electrophoresis. This methodology has been successfully utilized in studies of the M1

Page 44: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

30

muscarinic receptor114 and the OX1 orexin receptor115, among others, to probe the

oligomeric states of these GPCRs, as the minimal disruption of multiprotein complexes

with this approach allows for visualization of the fraction of receptors that exist as

monomers vs dimers and higher order oligomers, a distinct advantage over conventional

Co-IP approaches. Additionally, the individual components of the multiprotein

complexes visualized through this approach can be subsequently dissected through

conventional SDS-PAGE, revealing the constituent proteins of a putative oligomer116.

This approach, similar to Co-IP, still requires receptors be engineered to possess an

epitope tag, and cannot be utilized to assess dynamic aspects of GPCR dimerization, such

as dimer half-life and ontogeny. Still, BN-PAGE utilizes a more streamlined

methodology than Co-IP analysis and it remains a valuable tool in probing GPCR dimers

and oligomers.

There are significant drawbacks for each of the approaches listed above. First,

both methodologies require cells to be lysed and harvested, and thus cannot be employed

for investigations of GPCR dimerization dynamics in living cells. Additionally, in many

cases, receptors must be overexpressed in order to be appropriately resolved through

Western blot analysis, raising the possibility that these assays detect interactions between

receptors that are not physiologically relevant112. Also, as these methodologies do not

distinguish between direct receptor-receptor interactions and their incorporation in larger

protein complexes, these methodologies are often complemented by microscopy-based

approaches to further verify their findings.

Page 45: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

31

Microscopy-based approaches to resolve GPCR dimerization

A number of microscopic techniques have been adapted to probe the existence

and organization of putative GPCR oligomers. Historically, the most common technique

utilized in this regard has been Fluorescent Resonance Energy Transfer (FRET). This

methodology, first proposed by Theodor Förster in 1948, involves energy transfer

between complementary fluorophores through dipole-dipole interactions, with one

fluorophore acting as an energy donor and another acting as an energy acceptor117. For

this energy transfer to occur, both fluorophores must be in close proximity to one another,

with the upper limit for detection of FRET being ~100 Å distance between the two

fluorophores involved. Additionally, the excitation spectrum for the acceptor fluorophore

must overlap with the emission spectrum for the donor fluorophore, even though the

energy transfer between molecules does not rely on emitted photons from the donor

fluorophore118. Furthermore, while not a specific requirement for FRET, the emission

spectra of the fluorophores utilized must have sufficient separation to allow for the

resolution of one fluorophore from the other when visualized. Common fluorophore pairs

utilized in FRET analysis (listed as donor/acceptor) are Cyan Fluorescent Protein (CFP)/

Yellow Fluorescent Protein (YFP), Cerulean/Venus, and Cy3/Cy5119. Multiple methods

to determine FRET efficiency, or the fraction of donor fluorophores that transfer energy

to acceptor molecules, exist, including time resolved FRET (TR-FRET), acceptor

photobleaching, and sensitized emission120. Sensitized emission FRET, perhaps the

simplest of these methodologies, involves co-expressing donor and acceptor-tagged

proteins in the same cell and exciting only the donor fluorophore. Under ideal conditions

(no cross-talk between fluorescent proteins), any subsequent emission from the acceptor

Page 46: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

32

fluorophore would be the direct result of non-radiative energy transfer from the donor to

the acceptor molecule. FRET efficiency can be determined by comparing acceptor

fluorescence in co-transfected cells with the fluorescent spectra of cells expressing only

the donor or acceptor tagged molecule. This method, while straightforward, suffers from

complications due to the significant cross-talk exhibited by most FRET pair fluorescent

proteins, and as such, requires multiple filter combinations and post-processing

corrections to be employed to accurately analyze FRET efficiencies.

A more robust quantitative FRET methodology is acceptor photobleaching FRET.

This methodology relies on the fact that, if a FRET response is occurring between a

donor and acceptor fluorophore, the donor emission spectra is “quenched” by non-

radiative energy transfer to the acceptor fluorophore. Following initial image acquisition,

the acceptor fluorophore is photobleached by high intensity laser pulses, eliminating

energy transfer between the donor and acceptor fluorophores, which can be visualized as

an increase in donor fluorescence following acceptor photobleaching120. This

methodology eliminates many issues involving cross talk between fluorophores, as long

as the donor fluorophore is not bleached by the acceptor photobleaching pulse, as the

increase in donor fluorescence is directly proportional to the fraction of donor molecules

transferring energy to acceptor fluorophores, which is useful when quantifying

differential FRET responses.

Time resolved FRET (TR-FRET) is another methodology that seeks to maximize

signal to noise ratios by using lanthanide fluorophores for both donor and acceptor

molecules121. In contrast to GFP derived fluorophores, which possess fluorescent

lifetimes ranging from approximately 2-4 nanoseconds, lanthanide fluorophores possess

Page 47: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

33

much longer fluorescent lifetimes (~1 millisecond), which allows for a delay of

approximately 50 microseconds to be added between donor excitation and emission

signal acquisition121. During this delay, any background autofluorescence from tissue will

dissipate, resulting in increased signal to noise ratios in TR-FRET data when compared to

other FRET methodologies. The large distances between peak excitation and emission

wavelengths in the lanthanide fluorophores used in TR-FRET studies also minimizes

cross-talk and bleedthrough between the donor and acceptor fluorophores, further

increasing signal to noise ratios with this methodology122. As such, TR-FRET remains a

useful and robust methodology for determining the oligomeric state of GPCRs.

All FRET methodologies suffer from inherent and methodological pitfalls. For

example, intermolecular FRET efficiency is directly dependent on the stoichiometry of

the donor and acceptor-tagged proteins of interest. As such, many FRET studies bias the

likelihood of donor-acceptor interactions by transfecting a greater ratio of acceptor-

tagged receptors than donor-tagged receptors. This increases the likelihood of donor-

tagged proteins interacting with acceptor tagged proteins at the cell surface, increasing

observed FRET efficiencies. However, the stochastic incorporation of plasmids inherent

to transient transfections can lead to large cell to cell variance in observed FRET

efficiencies. Additionally, FRET relies on the three-dimensional positioning and

orientation of the fluorophores involved, and as such, the location of the fluorophore

fusion with the receptor molecule, and any interactions, such as ligand binding, that

impact the conformation of the receptor can potentially influence observed FRET

efficiencies without reflecting a change in receptor-receptor interactions. FRET

efficiencies are also impacted by membrane curvature and overall receptor density,

Page 48: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

34

calling into question the results of FRET studies which rely on overexpression of tagged

receptors in a heterologous expression system123. As such, FRET studies are often paired

with biochemical assays, such as Co-IP, to increase confidence in their findings.

A similar methodology to FRET commonly used to probe the oligomeric states of

various GPCRs is bioluminescent resonance energy transfer (BRET). This methodology

involves tagging of the donor molecule with a luciferase variant, which will typically

emit ~480nm photons following addition of a luciferin substrate. This emitted photon is

capable of exciting a fluorophore on the acceptor molecule, typically a GFP or YFP

variant, resulting in subsequent photon emission between 510-530nm depending on the

acceptor fluorophore used124. Similar to FRET, the donor luciferase molecule must be

within 100 Å of the acceptor fluorophore for BRET interactions to occur. One specific

advantage of this methodology over conventional FRET methods is the separation of

emission spectra between donor and acceptor molecules, increasing signal to noise

ratio125. Additionally, BRET does not rely on laser stimulation of a donor fluorophore

molecule, greatly reducing background autofluorescence and further enhancing signal

acquisition125. However, BRET suffers from similar complications as FRET analysis,

being highly dependent on receptor density, with receptor overexpression capable of

greatly increasing observed BRET responses121. Also, BRET signals are difficult to

detect at low receptor expression levels, limiting its value in studying receptor-receptor

interactions in native tissues121.

An additional microscopy-based approach to resolve GPCR-GPCR interactions is

biomolecular fluorescent complementation, or BiFC. This methodology involves the

generation of tagged acceptor and donor receptors with complementary fragments of a

Page 49: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

35

fluorophore molecule, most commonly a GFP-derived fluorophore126. When expressed

alone, the fluorophore fragments attached to either the donor or acceptor receptor are

unable to adopt a conformation capable of producing fluorescence. However, co-

localization of the complementary fluorophore fragments within approximately 7 nm

leads to a functional reconstitution of the split fluorophore molecule, which can be

visualized through fluorescent microscopy127. A specific advantage of this methodology

over both BRET and FRET approaches in the ability to resolve BiFC interactions at low

receptor densities that may more accurately reflect physiological expression levels than

receptor overexpression commonly seen in both BRET and FRET investigations128.

Additionally, BiFC does not suffer from issues such as crosstalk or bleedthrough between

acceptor and donor fluorophores, as is common with FRET investigations. This does not

mean that BiFC is without its flaws. One major issue regarding BiFC analysis of

receptor-receptor interactions is that fluorescent complementation of acceptor and donor

fragments may occur as a result of “kiss and run” interactions between donor and

acceptor molecules, rather than reflecting stable interactions between the receptors

involved. This also renders this methodology incapable of studying dynamic protein-

protein interactions, as fluorescent complementation is a largely irreversible event129.

These concerns, similar to BRET and FRET based investigations, result in receptor-

receptor interactions suggested by BiFC being further verified by additional

methodologies, such as Co-IP, to increase confidence in BiFC results129. Thus, similar to

other microscopy-based approaches, BiFC offers specific advantages and disadvantages

to resolving GPCR-GPCR interactions within the cell. Altogether, multiple microscopy-

based methodologies to resolve GPCR dimers exist, each of which offering specific

Page 50: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

36

benefits and tradeoffs in contrast to others, leading to complementary methodologies such

as co-immunoprecipitation to be utilized to increase confidence in FRET, BRET, and

BiFC studies.

Conclusion

Since the initial cloning of the β2AR nearly four decades ago, much progress has

been made towards a comprehensive understanding of GPCR function. It is now clear

that this receptor superfamily exhibits greater complexity in activation and signaling than

can be predicted by the classical two-state model. Additionally, as the number of

annotated GPCR dimers and oligomers continues to grow, the question of whether a

monomer or higher order structure represents the functional receptor unit for many

GPCRs remains the subject of much debate. Furthermore, receptor-receptor interactions

within a dimer can giving lead to transactivation or transdesensitization, which can

complicate efforts to assign specific downstream signaling pathways to individual

GPCRs. As such, continued investigation of conserved GPCR sequence motifs, as well as

allosteric modulators of GPCR signaling, may elucidate fundamental molecular

interactions and events underlying GPCR function and activity and further our

understanding of this protein superfamily.

Page 51: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

37

REFERENCES

1. Krishnan, A., Almén, M. S., Fredriksson, R. & Schiöth, H. B. The Origin of

GPCRs: Identification of Mammalian like Rhodopsin, Adhesion, Glutamate and

Frizzled GPCRs in Fungi. PLoS One 7, e29817 (2012).

2. Hanlon, C. D. & Andrew, D. J. Outside-in signaling – a brief review of GPCR

signaling with a focus on the Drosophila GPCR family. J Cell Sci 128, 3533–3542

(2015).

3. Hu, G.-M., Mai, T.-L. & Chen, C.-M. Visualizing the GPCR Network:

Classification and Evolution. Sci. Rep. 7, 15495 (2017).

4. Dixon, R. A. F. et al. Cloning of the gene and cDNA for mammalian β-adrenergic

receptor and homology with rhodopsin. Nature 321, 75–79 (1986).

5. Thomsen, W., Frazer, J. & Unett, D. Functional assays for screening GPCR

targets. Curr. Opin. Biotechnol. 16, 655–665 (2005).

6. Ferré, S. et al. G Protein-Coupled Receptor Oligomerization Revisited: Functional

and Pharmacological Perspectives. doi:10.1124/pr.113.008052.

7. Xu, J. et al. GPR68 Senses Flow and Is Essential for Vascular Physiology Article

GPR68 Senses Flow and Is Essential for Vascular Physiology. Cell 173, 762-

767.e16 (2018).

8. S-H Park, P., Lodowski, D. T. & Palczewski, K. Activation of G Protein-Coupled

Receptors: Beyond Two-State Models and Tertiary Conformational Changes

OVERVIEW OF G PROTEIN-COUPLED RECEPTOR STRUCTURE.

Page 52: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

38

doi:10.1146/annurev.pharmtox.48.113006.094630.

9. Bray, P. et al. Human cDNA clones for four species of G alpha s signal

transduction protein. Proc. Natl. Acad. Sci. U. S. A. 83, 8893–7 (1986).

10. Ahn, S. et al. A dominant-negative inhibitor of CREB reveals that it is a general

mediator of stimulus-dependent transcription of c-fos. Mol. Cell. Biol. 18, 967–77

(1998).

11. Lim, C. et al. Functional role of CREB-binding protein in the circadian clock

system of Drosophila melanogaster. Mol. Cell. Biol. 27, 4876–90 (2007).

12. Obel, L. F. et al. Brain glycogen—new perspectives on its metabolic function and

regulation at the subcellular level. Front. Neuroenergetics 4, 3 (2012).

13. Ichiki, T. Role of cAMP Response Element Binding Protein in Cardiovascular

Remodeling Good, Bad, or Both? (2006)

doi:10.1161/01.ATV.0000196747.79349.d1.

14. Alig, J. et al. Control of heart rate by cAMP sensitivity of HCN channels. Proc.

Natl. Acad. Sci. 106, 12189–12194 (2009).

15. Morgan, P. J. & Mordue, W. Cyclic AMP and locust diuretic hormone action.

Hormone induced changes in cAMP levels offers a novel method for detecting

biological activity of uncharacterized peptide. Insect Biochem. 15, 247–257

(1985).

16. Kandel, E. R. The molecular biology of memory: cAMP, PKA, CRE, CREB-1,

CREB-2, and CPEB. http://www.molecularbrain.com/content/5/1/14 (2012)

Page 53: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

39

doi:10.1186/1756-6606-5-14.

17. Sands, W. A., Woolson, H. D., Milne, G. R., Rutherford, C. & Palmer, T. M.

Exchange protein activated by cyclic AMP (Epac)-mediated induction of

suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells. Mol.

Cell. Biol. 26, 6333–46 (2006).

18. Taussig, R. & Gilman, A. G. Mammalian membrane-bound adenylyl cyclases. J.

Biol. Chem. 270, 1–4 (1995).

19. Padgett, C. L. & Slesinger, P. A. GABAB Receptor Coupling to G-proteins and

Ion Channels. Adv. Pharmacol. 58, 123–147 (2010).

20. Iannacone, M. J. et al. The RFamide receptor DMSR-1 regulates stress-induced

sleep in C. elegans. Elife 6, (2017).

21. Harden, T. K., Waldo, G. L., Hicks, S. N. & Sondek, J. Mechanism of activation

and inactivation of Gq/phospholipase C-β signaling nodes. Chem. Rev. 111, 6120–

9 (2011).

22. Heydorn, A. et al. Identification of a novel site within G protein alpha subunits

important for specificity of receptor-G protein interaction. Mol. Pharmacol. 66,

250–9 (2004).

23. Goldsmith, Z. G. & Dhanasekaran, D. N. G Protein regulation of MAPK networks.

Oncogene vol. 26 3122–3142 (2007).

24. Liu, K. peing et al. Calcium receptor-induced serotonin secretion by parafollicular

cells: Role of phosphatidylinositol 3-kinase-dependent signal transduction

Page 54: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

40

pathways. J. Neurosci. 23, 2049–2057 (2003).

25. Gough, N. R. Connecting GPCRs to NF-κB. Sci. STKE 2007, tw12–tw12 (2007).

26. K-K Fung, B. Characterization of Transducin from Bovine Retinal Rod Outer

Segments I. SEPARATION AND RECONSTITUTION OF THE SUBUNITS*. THE

JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A vol. 258

http://www.jbc.org/.

27. Spielman, A. I. Gustducin and its Role in Taste. J. Dent. Res. 77, 539–544 (1998).

28. Dascal, N. Ion-channel regulation by G proteins. Trends in Endocrinology and

Metabolism vol. 12 391–398 (2001).

29. Walsh, K. B. Targeting GIRK channels for the development of new therapeutic

agents. Front. Pharmacol. OCT, 64 (2011).

30. Snow, B. E. et al. A G protein γ subunit-like domain shared between RGS11 and

other RGS proteins specifies binding to Gβ5 subunits. Proc. Natl. Acad. Sci. 95,

13307–13312 (1998).

31. McPherson, K. B. et al. Regulators of G-Protein Signaling (RGS) Proteins

Promote Receptor Coupling to G-Protein-Coupled Inwardly Rectifying Potassium

(GIRK) Channels. J. Neurosci. 38, 8737–8744 (2018).

32. Taussig, R., Quarmby, L. M. & Gilman, A. G. THE JOURNAL OF BIOLOGICAL

CHEMISTRY Regulation of Purified Type I and Type I1 Adenylylcyclases by G

Protein Br Subunits* Plasmid Construction-To facilitate purification of type I

adeny-lylcyclase, we constructed a recombinant baculovims encoding a type. vol.

Page 55: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

41

268 http://www.jbc.org/content/268/1/9.full.pdf (1993).

33. Litosch, I. G-protein inhibition of phospholipase C-beta 1 in membranes: role of

G-protein beta gamma subunits. Biochem. J. 319 ( Pt 1), 173–8 (1996).

34. Wu, D., Katz, A. & Simon, M. I. Activation of phospholipase C beta 2 by the

alpha and beta gamma subunits of trimeric GTP-binding protein. Proc. Natl. Acad.

Sci. U. S. A. 90, 5297–301 (1993).

35. Gurevich, V. V & Gurevich, E. V. GPCR Signaling Regulation: The Role of

GRKs and Arrestins. Front. Pharmacol. 10, 125 (2019).

36. Palczewski, K. & Saari, J. C. Activation and inactivation steps in the visual

transduction pathway. Curr. Opin. Neurobiol. 7, 500–504 (1997).

37. Smith, J. S. & Rajagopal, S. The β-Arrestins: Multifunctional Regulators of G

Protein-coupled Receptors. J. Biol. Chem. 291, 8969–77 (2016).

38. Niu, S. et al. Beta-Arrestin 1 Mediates Liver Thyrotropin Regulation of

Cholesterol Conversion Metabolism via the Akt-Dependent Pathway. Int. J.

Endocrinol. 2018, 1–12 (2018).

39. Donthamsetti, P. et al. Arrestin recruitment to dopamine D2 receptor mediates

locomotion but not incentive motivation. Mol. Psychiatry 1 (2018)

doi:10.1038/s41380-018-0212-4.

40. Bockaert, J., Fagni, L., Dumuis, A. & Marin, P. GPCR interacting proteins (GIP).

Pharmacol. Ther. 103, 203–221 (2004).

41. Dickerson, I. M. Role of CGRP-receptor component protein (RCP) in CLR/RAMP

Page 56: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

42

function. Curr. Protein Pept. Sci. 14, 407–15 (2013).

42. McLatchie, L. M. et al. RAMPs regulate the transport and ligand specificity of the

calcitonin-receptor-like receptor. Nature 393, 333–339 (1998).

43. Martin, A. L., Steurer, M. A. & Aronstam, R. S. Constitutive Activity among

Orphan Class-A G Protein Coupled Receptors. PLoS One 10, e0138463 (2015).

44. Seifert, R. & Wenzel-Seifert, K. Constitutive activity of G-protein-coupled

receptors: cause of disease and common property of wild-type receptors. Naunyn.

Schmiedebergs. Arch. Pharmacol. 366, 381–416 (2002).

45. Kobilka, B. K. & Deupi, X. Conformational complexity of G-protein-coupled

receptors. Trends Pharmacol. Sci. 28, 397–406 (2007).

46. Liu, F. et al. Involvement of both G(q/11) and G(s) proteins in gonadotropin-

releasing hormone receptor-mediated signaling in L beta T2 cells. J. Biol. Chem.

277, 32099–108 (2002).

47. Harmar, A. J. et al. cDNA sequence of human β-preprotachykinin, the common

precursor to substance P and neurokinin A. FEBS Lett. 208, 67–72 (1986).

48. Palanche, T. et al. The Neurokinin A Receptor Activates Calcium and cAMP

Responses through Distinct Conformational States. J. Biol. Chem. 276, 34853–

34861 (2001).

49. Wacker, D., Stevens, R. C. & Roth, B. L. How Ligands Illuminate GPCR

Molecular Pharmacology. Cell vol. 170 414–427 (2017).

50. Ballesteros, J. A. et al. Activation of the β2-Adrenergic Receptor Involves

Page 57: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

43

Disruption of an Ionic Lock between the Cytoplasmic Ends of Transmembrane

Segments 3 and 6. J. Biol. Chem. 276, 29171–29177 (2001).

51. Pruitt, M. M., Lamm, M. H. & Coffman, C. R. Molecular dynamics simulations on

the Tre1 G protein-coupled receptor: exploring the role of the arginine of the NRY

motif in Tre1 structure. BMC Struct. Biol. 13, 15 (2013).

52. Ballesteros, J. A. et al. Activation of the beta 2-adrenergic receptor involves

disruption of an ionic lock between the cytoplasmic ends of transmembrane

segments 3 and 6. J. Biol. Chem. 276, 29171–7 (2001).

53. Bhattacharya, S., Salomon-Ferrer, R., Lee, S. & Vaidehi, N. Conserved

Mechanism of Conformational Stability and Dynamics in G-Protein-Coupled

Receptors. J. Chem. Theory Comput. 12, 5575–5584 (2016).

54. Rovati, G. E., Rie Capra, V. & Neubig, R. R. The Highly Conserved DRY Motif

of Class A G Protein-Coupled Receptors: Beyond the Ground State. Mol.

Pharmacol. 71, 959–964 (2007).

55. Rasmussen, S. G. F. et al. Mutation of a Highly Conserved Aspartic Acid in the β

2 Adrenergic Receptor: Constitutive Activation, Structural Instability, and

Conformational Rearrangement of Transmembrane Segment 6. Mol. Pharmacol.

56, 175–184 (2018).

56. Alewijnse, A. E. et al. The effect of mutations in the DRY motif on the

constitutive activity and structural instability of the histamine H(2) receptor. Mol.

Pharmacol. 57, 890–898 (2000).

Page 58: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

44

57. Vogel, R. et al. Functional Role of the “Ionic Lock”—An Interhelical Hydrogen-

Bond Network in Family A Heptahelical Receptors. J. Mol. Biol. 380, 648–655

(2008).

58. Rosenthal, W., Antaramian, A., Gilbert, S. & Birnbaumer, M. Nephrogenic

diabetes insipidus. A V2 vasopressin receptor unable to stimulate adenylyl cyclase.

Journal of Biological Chemistry vol. 268

http://www.jbc.org/content/268/18/13030.full.pdf (1993).

59. Costa, E. M. F. et al. Two Novel Mutations in the Gonadotropin-Releasing

Hormone Receptor Gene in Brazilian Patients with Hypogonadotropic

Hypogonadism and Normal Olfaction 1. J. Clin. Endocrinol. Metab. 86, 2680–

2686 (2001).

60. Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. & Johnson, E. C. Unexpected

role of a conserved domain in the first extracellular loop in G protein-coupled

receptor trafficking. Biochem. Biophys. Res. Commun. 503, 1919–1926 (2018).

61. Klco, J. M., Nikiforovich, G. V & Baranski, T. J. Genetic Analysis of the First and

Third Extracellular Loops of the C5a Receptor Reveals an Essential WXFG Motif

in the First Loop. J. Biol. Chem. 281, 12010–12019 (2006).

62. Hulme, E. C. GPCR activation: a mutagenic spotlight on crystal structures. Trends

Pharmacol. Sci. 34, 67–84 (2013).

63. De Filippo, E. et al. Role of extracellular cysteine residues in the adenosine A2A

receptor. Purinergic Signal. 12, 313–329 (2016).

Page 59: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

45

64. Olivella, M., Caltabiano, G. & Cordomí, A. The role of Cysteine 6.47 in class A

GPCRs. BMC Struct. Biol. 13, 3 (2013).

65. Weis, W. I. & Kobilka, B. K. The Molecular Basis of G Protein–Coupled Receptor

Activation. Annu. Rev. Biochem. 87, 897–919 (2018).

66. Shi, L. et al. Modulation of the proline kink in transmembrane 6 by a rotamer

toggle switch. J. Biol. Chem. 277, 40989–96 (2002).

67. Biebermann, H. et al. New Pathogenic Thyrotropin Receptor Mutations Decipher

Differentiated Activity Switching at a Conserved Helix 6 Motif of Family A

GPCR. J. Clin. Endocrinol. Metab. 97, E228–E232 (2012).

68. Standfuss, J. et al. The structural basis of agonist-induced activation in

constitutively active rhodopsin. Nature 471, 656–60 (2011).

69. Cherezov, V. et al. High-resolution crystal structure of an engineered human

beta2-adrenergic G protein-coupled receptor. Science 318, 1258–65 (2007).

70. Katritch, V., Cherezov, V. & Stevens, R. C. Structure-Function of the G Protein–

Coupled Receptor Superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556

(2013).

71. Barak, L. S. et al. A highly conserved tyrosine residue in G protein-coupled

receptors is required for agonist-mediated β2-adrenergic receptor sequestration. J.

Biol. Chem. 269, 2790–2795 (1994).

72. Barak, L. S., Ménard, L., Ferguson, S. S. G., Colapietro, A. M. & Caron, M. G.

The Conserved Seven-Transmembrane Sequence NP(X)2,3Y of the G-Protein-

Page 60: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

46

Coupled Receptor Superfamily Regulates Multiple Properties of the β2-Adrenergic

Receptor. Biochemistry 34, 15407–15414 (1995).

73. Kalatskaya, I. et al. Mutation of tyrosine in the conserved NPXXY sequence leads

to constitutive phosphorylation and internalization, but not signaling, of the human

B2 bradykinin receptor. J. Biol. Chem. 279, 31268–76 (2004).

74. Slice, L. W. et al. The conserved NPX(n)Y motif present in the gastrin-releasing

peptide receptor is not a general sequestration sequence. J. Biol. Chem. 269,

21755–21761 (1994).

75. Rosenbaum, D. M., Rasmussen, S. G. F. & Kobilka, B. K. The structure and

function of G-protein-coupled receptors. Nature 459, 356–363 (2009).

76. Ragnarsson, L., Andersson, Å., Thomas, W. G. & Lewis, R. J. Mutations in the

NPxxY motif stabilize pharmacologically distinct conformational states of the

α1B- and β2-adrenoceptors. Sci. Signal. 12, eaas9485 (2019).

77. White, J. H. et al. Heterodimerization is required for the formation of a functional

GABA(B) receptor. Nature 396, 679–682 (1998).

78. Margeta-Mitrovic, M., Jan, Y. N. & Jan, L. Y. A Trafficking Checkpoint Controls

GABAB Receptor Heterodimerization. Neuron 27, 97–106 (2000).

79. Chun, L., Zhang, W. & Liu, J. Structure and ligand recognition of class C GPCRs.

Acta Pharmacol. Sin. 33, 312–323 (2012).

80. Grushevskyi, E. O. et al. Stepwise activation of a class C GPCR begins with

millisecond dimer rearrangement. Proc. Natl. Acad. Sci. U. S. A. 116, 10150–

Page 61: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

47

10155 (2019).

81. Møller, T. C., Moreno-Delgado, D., Pin, J.-P. & Kniazeff, J. Class C G protein-

coupled receptors: reviving old couples with new partners. Biophys. Reports 3, 57–

63 (2017).

82. Milligan, G., Ward, R. J. & Marsango, S. GPCR homo-oligomerization. Current

Opinion in Cell Biology vol. 57 40–47 (2019).

83. Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and

cyclic peptide antagonists. Science 330, 1066–71 (2010).

84. Mancia, F., Assur, Z., Herman, A. G., Siegel, R. & Hendrickson, W. A. Ligand

sensitivity in dimeric associations of the serotonin 5HT2c receptor. EMBO Rep.

(2008) doi:10.1038/embor.2008.27.

85. Borroto-Escuela, D. O. et al. Mapping the interface of a GPCR Dimer: A structural

model of the A2A Adenosine and D2 dopamine receptor heteromer. Front.

Pharmacol. (2018) doi:10.3389/fphar.2018.00829.

86. Fotiadis, D. et al. The G protein-coupled receptor rhodopsin in the native

membrane. FEBS Lett. 564, 281–288 (2004).

87. Petersen, J. et al. Agonist-induced dimer dissociation as a macromolecular step in

G protein-coupled receptor signaling. Nat. Commun. 8, 226 (2017).

88. Wolf, S. & Grünewald, S. Sequence, Structure and Ligand Binding Evolution of

Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based

Phylogenetic Analysis. PLoS One 10, e0123533 (2015).

Page 62: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

48

89. Huang, J., Chen, S., Zhang, J. J. & Huang, X. Y. Crystal structure of oligomeric β

1-adrenergic G protein-coupled receptors in ligand-free basal state. Nat. Struct.

Mol. Biol. (2013) doi:10.1038/nsmb.2504.

90. Ward, R. J., Pediani, J. D., Harikumar, K. G., Miller, L. J. & Milligan, G. Spatial

intensity distribution analysis quantifies the extent and regulation of

homodimerization of the secretin receptor. Biochem. J. 474, 1879–1895 (2017).

91. Harikumar, K. G., Ball, A. M., Sexton, P. M. & Miller, L. J. Importance of lipid-

exposed residues in transmembrane segment four for family B calcitonin receptor

homo-dimerization. Regul. Pept. 164, 113–119 (2010).

92. May, L. T., Bridge, L. J., Stoddart, L. A., Briddon, S. J. & Hill, S. J. Allosteric

interactions across native adenosine-A 3 receptor homodimers: Quantification

using single-cell ligand-binding kinetics. FASEB J. 25, 3465–3476 (2011).

93. Urizar, E. et al. Glycoprotein hormone receptors: Link between receptor

homodimerization and negative cooperativity. EMBO J. 24, 1954–1964 (2005).

94. El-Asmar, L. et al. Evidence for negative binding cooperativity within CCR5-

CCR2b heterodimers. Mol. Pharmacol. 67, 460–9 (2005).

95. Albizu, L. et al. Time-resolved FRET between GPCR ligands reveals oligomers in

native tissues. Nat. Chem. Biol. 6, 587–594 (2010).

96. Ferré, S. et al. G Protein–Coupled Receptor Oligomerization Revisited: Functional

and Pharmacological Perspectives. Pharmacol. Rev. 66, 413 LP – 434 (2014).

97. Vischer, H. F., Castro, M. & Pin, J. P. G protein-coupled receptor multimers: A

Page 63: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

49

question still open despite the use of novel approaches. Mol. Pharmacol. (2015)

doi:10.1124/mol.115.099440.

98. Zhao, G. Q. et al. The Receptors for Mammalian Sweet and Umami Taste. Cell

115, 255–266 (2003).

99. El Moustaine, D. et al. Distinct roles of metabotropic glutamate receptor

dimerization in agonist activation and G-protein coupling. Proc. Natl. Acad. Sci.

U. S. A. 109, 16342–7 (2012).

100. Pioszak, A. A., Harikumar, K. G., Parker, N. R., Miller, L. J. & Xu, H. E. Dimeric

arrangement of the parathyroid hormone receptor and a structural mechanism for

ligand-induced dissociation. J. Biol. Chem. 285, 12435–44 (2010).

101. Robinson, P. J., Pringle, M. A., Woolhead, C. A. & Bulleid, N. J. Folding of a

single domain protein entering the endoplasmic reticulum precedes disulfide

formation. J. Biol. Chem. (2017) doi:10.1074/jbc.M117.780742.

102. Dijkman, P. M. et al. Dynamic tuneable G protein-coupled receptor monomer-

dimer populations. Nat. Commun. (2018) doi:10.1038/s41467-018-03727-6.

103. Herrick-Davis, K., Weaver, B. A., Grinde, E. & Mazurkiewicz, J. E. Serotonin 5-

HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time

visualization with confocal fluorescence resonance energy transfer. J. Biol. Chem.

281, 27109–16 (2006).

104. King, K., Dohlman, H. G., Thorner, J., Caron, M. G. & Lefkowitz, R. J. Control of

yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and

Page 64: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

50

Gs alpha subunit. Science 250, 121–3 (1990).

105. Tamma, G., Carmosino, M., Svelto, M. & Valenti, G. Bradykinin signaling

counteracts cAMP-elicited aquaporin 2 translocation in renal cells. J. Am. Soc.

Nephrol. (2005) doi:10.1681/ASN.2005020190.

106. Haack, K. K. V et al. A novel bioassay for detecting GPCR heterodimerization:

Transactivation of beta 2 adrenergic receptor by bradykinin receptor. J. Biomol.

Screen. (2010) doi:10.1177/1087057109360254.

107. Rivero-Müller, A. et al. Rescue of defective G protein - Coupled receptor function

in vivo by intermolecular cooperation. Proc. Natl. Acad. Sci. U. S. A. (2010)

doi:10.1073/pnas.0906695106.

108. Alonso, N. et al. Cross-desensitization and cointernalization of H1 and H2

histamine receptors reveal new insights into histamine signal integration. Mol.

Pharmacol. (2013) doi:10.1124/mol.112.083394.

109. Sakai, T. et al. Evidence for differential regulation of GnRH signaling via

heterodimerization among GnRH receptor paralogs in the protochordate, Ciona

intestinalis. Endocrinology 153, 1841–1849 (2012).

110. González-Maeso, J. et al. Identification of a serotonin/glutamate receptor complex

implicated in psychosis. Nature 452, 93–97 (2008).

111. Moreno, J. L., Holloway, T., Albizu, L., Sealfon, S. C. & González-Maeso, J.

Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and

behavioral effects induced by hallucinogenic 5-HT2A receptor agonists. Neurosci.

Page 65: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

51

Lett. 493, 76–9 (2011).

112. Szidonya, L., Cserzo, M. & Hunyady, L. Dimerization and oligomerization of G-

protein-coupled receptors: Debated structures with established and emerging

functions. Journal of Endocrinology (2008) doi:10.1677/JOE-07-0573.

113. Schägger, H. & von Jagow, G. Blue native electrophoresis for isolation of

membrane protein complexes in enzymatically active form. Anal. Biochem. 199,

223–231 (1991).

114. Marquer, C. et al. Influence of MT7 toxin on the oligomerization state of the M1

muscarinic receptor1. Biol. Cell (2010) doi:10.1042/bc20090171.

115. Xu, T. R., Ward, R. J., Pediani, J. D. & Milligan, G. The orexin OX 1 receptor

exists predominantly as a homodimer in the basal state: Potential regulation of

receptor organization by both agonist and antagonist ligands. Biochem. J. (2011)

doi:10.1042/BJ20110230.

116. Fiala, G. J., Schamel, W. W. A. & Blumenthal, B. Blue native polyacrylamide gel

electrophoresis (BN-PAGE) for analysis of multiprotein complexes from cellular

lysates. J. Vis. Exp. (2010) doi:10.3791/2164.

117. Förster, T. Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Phys.

437, 55–75 (1948).

118. Sekar, R. B. & Periasamy, A. Fluorescence resonance energy transfer (FRET)

microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629–33

(2003).

Page 66: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

52

119. Shrestha, D., Jenei, A., Nagy, P., Vereb, G. & Szöllősi, J. Understanding FRET as

a Research Tool for Cellular Studies. Int. J. Mol. Sci. 16, 6718 (2015).

120. Piston, D. W. & Kremers, G.-J. Fluorescent protein FRET: the good, the bad and

the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

121. Cottet, M. et al. BRET and Time-resolved FRET strategy to study GPCR

oligomerization: from cell lines toward native tissues. Front. Endocrinol.

(Lausanne). 3, 92 (2012).

122. Bazin, H., Trinquet, E. & Mathis, G. Time resolved amplification of cryptate

emission: a versatile technology to trace biomolecular interactions. Rev. Mol.

Biotechnol. 82, 233–250 (2002).

123. Walsh, S. M. et al. Single Proteoliposome High-Content Analysis Reveals

Differences in the Homo-Oligomerization of GPCRs. Biophys. J. 115, 300–312

(2018).

124. Kocan, M. & Pfleger, K. D. G. Study of GPCR-protein interactions by BRET.

Methods Mol. Biol. 746, 357–371 (2011).

125. Salahpour, A. et al. BRET biosensors to study GPCR biology, pharmacology, and

signal transduction. Front. Endocrinol. (Lausanne). 3, 105 (2012).

126. Vidi, P.-A., Przybyla, J. A., Hu, C.-D. & Watts, V. J. Visualization of G protein-

coupled receptor (GPCR) interactions in living cells using bimolecular

fluorescence complementation (BiFC). Curr. Protoc. Neurosci. Chapter 5, Unit

5.29 (2010).

Page 67: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

53

127. Berendzen, K. et al. Screening for in planta protein-protein interactions combining

bimolecular fluorescence complementation with flow cytometry. Plant Methods 8,

25 (2012).

128. Kerppola, T. K. Bimolecular Fluorescence Complementation (BiFC) Analysis as a

Probe of Protein Interactions in Living Cells. Annu. Rev. Biophys. 37, 465–487

(2008).

129. Miller, K. E., Kim, Y., Huh, W.-K. & Park, H.-O. Bimolecular Fluorescence

Complementation (BiFC) Analysis: Advances and Recent Applications for

Genome-Wide Interaction Studies. J. Mol. Biol. 427, 2039–2055 (2015).

Page 68: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

54

Figure 1: Two-state model of GPCR activation. In the inactive receptor state (Fig. 1A),

in the absence of ligand, the receptor remains bound to an intracellular heterotrimeric G

protein. The α subunit in the heterotrimeric G protein is bound to GDP and is inactive.

Upon ligand binding (Fig. 1B), the receptor undergoes a conformational change, allowing

it to function as a guanine nucleotide exchange factor (GEF), facilitating the association

of a GTP molecule with the associated α subunit. This event activates the α subunit and

associated βγ subunits, leading to the dissociation of the heterotrimeric G protein from

the activated receptor, where they can then signal through a variety of intracellular

pathways.

Page 69: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

55

Figure 2: Functional importance of GPCR heterodimerization. In a classical,

monomeric receptor model of GPCR function, receptor A and receptor B signal through

disparate pathways independent of one another (Fig. 2A). In the case of

heterodimerization, ligand binding to receptor B can lead to intracellular signaling

through receptor A, a phenomenon referred to as transactivation (Fig. 2B).

Heterodimerization can also lead to transdesensitization (Fig. 2C), where ligand binding

to receptor B leads to β-arrestin association and subsequent internalization of the AB

receptor complex. This causes desensitization of receptor A in the absence of ligand

binding to this receptor.

Page 70: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

56

CHAPTER II: Unexpected role of a conserved domain in extracellular loop 1 in G

protein coupled receptor trafficking

Michael J. Rizzo1, Jack P. Evans1, Morgan Burt1, Erik C. Johnson1,2*

1 Department of Biology, Wake Forest University, Winston-Salem, NC 27109

2 and Center for Molecular Signaling

*Author for Correspondence: [email protected]

Keywords: G protein-coupled receptor (GPCR), membrane trafficking, signaling,

membrane transport, mutagenesis

The work contained in this chapter was initially published in the journal Biochemical and

Biophysical Research Communications. M.J. Rizzo, J.P. Evans, M. Burt, C.J. Saunders,

E.C. Johnson, “Unexpected role of a conserved domain in the first extracellular loop in G

protein-coupled receptor trafficking”, Biochem. Biophys. Res. Commun. 503 (2018).

Experiments were conceived by MJ Rizzo and EC Johnson. Reagents were generated and

experiments were performed by MJ Rizzo, JP Evans, and M Burt. Data were analyzed by

MJ Rizzo and EC Johnson. The manuscript was drafted by MJ Rizzo and edited by EC

Johnson.

Page 71: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

57

ABSTRACT

G protein coupled receptors are the largest superfamily of cell surface receptors in

the metazoa and play critical roles in transducing extracellular signals into intracellular

responses. This action is mediated through a conformational change in the receptor

following ligand binding. A number of conserved motifs play critical roles in GPCR

function and stability, but a particular, highly conserved motif in extracellular loop

one (EL1) remains under investigated. This WxFG motif is present in ~90% of Class A

GPCRs and is prevalent in 17 of the 19 Class A GPCR subfamilies, yet its function

remains incompletely elucidated. Using site-directed mutagenesis, we mutagenized a

conserved tryptophan residue in the highly conserved WxFG motif in EL1 in eight

receptors from disparate class A GPCR subfamilies. We first targeted the Drosophila

leucokinin receptor and found that substitution of any non-aromatic amino acid for the

conserved tryptophan ablated receptor function. Additionally, tryptophan to leucine

substitutions in the follicle stimulating hormone receptor (FSHR), Galanin receptor

(GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic

acetylcholine receptor (mACHR1) lead to a loss of signaling response in each receptor.

We then utilized YFP tagged wild-type and mutant LKR, CRZR, and 5HT2cR receptors

to visualize these receptors in the cell and show that mutant receptor variants exhibited a

severe reduction in plasma membrane expression, indicating aberrant trafficking in these

receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR

trafficking and overall receptor function.

Page 72: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

58

INTRODUCTION

G protein coupled receptors (GPCRs) are the largest receptor superfamily present

throughout the metazoa1. Approximately 5% of all human genes encode these receptors

and these molecules are a target for approximately 50% of all extant drugs2. GPCRs play

a major role in myriad physiologies, including vision, taste, neurotransmission, hormonal

communication, and reproduction3. GPCRs transduce multiple disparate extracellular

signals into specific intracellular responses, most commonly increasing or inhibiting

intracellular calcium and cAMP and modulating gene expression4. Specifically, a

conformational change induced by ligand binding enables activation of intracellular

heterotrimeric G protein complexes, which in turn modulate disparate second

messengers.

Given the fundamental importance of GPCRs in a wide variety of behaviors and

physiologies, many structural-functional studies have aimed to understand the molecular

dynamics of receptor activity. Additionally, the crystal structure of rhodopsin has helped

identify a number of highly conserved motifs that have critical roles in wild type GPCR

function5. For example, various motifs have been implicated in activation (e.g., DRY,

CWxP), signal termination and receptor endocytosis (e.g., NPxxY), and endoplasmic

reticulum to cell surface trafficking (e.g., FX6LL)6–8. A particular motif that remains

relatively unexplored is the WxFG motif in extracellular loop one (EL1). This motif had

been initially described in the C5a receptor, wherein mutagenesis of the highly conserved

tryptophan residue led to a loss of signaling, presumably through a disruption of receptor

signaling downstream of ligand binding9. Subsequent studies suggested that this

tryptophan residue coevolved with proline residues on transmembrane domain 2 (TM2)

Page 73: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

59

and/or TM5, presumably to stabilize receptor conformation10. In this study, we first

investigated the prevalence of the WxFG motif in different receptor subfamilies and in

multiple taxa and established that this domain is widespread throughout Class A GPCRs.

We then assessed the functional roles of the tryptophan residue and found a critical role

for this residue for normal receptor function. We also found that this mutation ablated

constitutive signaling from a modified receptor. Evaluation of receptor distribution using

fluorescently tagged receptors revealed aberrant cellular localization, with the majority of

the mutant receptors restricted to internal membrane compartments. Structural modeling

of these receptor variants suggests this residue is critical for overall receptor topology.

Collectively, our results implicate that the WxFG motif plays a critical role in appropriate

GPCR cell surface trafficking and function.

Page 74: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

60

METHODS

Receptor sequence alignment:

We adopted the receptor subfamily A classification according to the phylogenetic

analysis of Joost and Methner to identify human receptors for each receptor subfamily11.

For a phylogenetic analysis, we used BLAST to search for homologous receptors to the

human subfamily receptors in Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio,

Ciona intestinalis, Drosophila melanogaster, and Caenorhabditis elegans. Only

identified and validated receptor types were included in further analysis. Receptor

sequences were then entered into TMHMM server to identify sequences corresponding to

the first extracellular loop and consensus motifs for receptor subfamilies were generated

using the Seq2LOGO webserver.

GPCR cloning and mutagenesis:

All GPCRs were cloned into a pcDNA3 expression vector. The Drosophila receptors

originated from amplification from cDNA libraries and mammalian receptors were

obtained from Addgene and cdna.org libraries. The human 5HT2c receptor was

generously donated by Dr. Katherine Herrick-Davis. Mutagenic primers were designed

targeting the tryptophan residue to alter it to a leucine or other amino acid. Site-directed

PCR mutagenesis was performed using both classical PCR mutagenesis or single primer

reactions in parallel (SPRINP) 12,13. The restriction enzyme, DpnI, was utilized to remove

template receptor molecules following PCR mutagenesis. All receptor sequences were

verified through ABI 3730XL sequencing through Eton Bioscience INC (Research

Triangle Park, NC).

Page 75: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

61

GPCR Signaling Assays:

Galanin, somatostatin, and FSH were purchased from Phoenix Pharmaceticals

(Burlingame, CA). Leukokinin and adipokinetic hormone peptides from Drosophila

were synthesized by Multiple Peptide Systems (San Diego, CA). Acetylcholine and

corazonin were purchased from Sigma Chemicals (St. Louis, MO). HEK-293T cells were

transfected with GPCR recombinant DNA and either a CRE-luciferase or SRE-luciferase

reporter DNA at a 5:1 ratio of receptor: reporter construct as previously described14. For

assessing signaling emanating from Gi coupled receptors, the Gα16 was transfected at a

2:1 ratio to receptor construct15. Following transfection, 96 well plates were seeded with

cells and incubated with vehicle (MEM) or ligand for four hours (100,000 cells per well,

3 wells per independent transfection, 9 wells per condition). Following incubation,

luciferase activity was assessed using the Steadylite plus Reporter Gene Assay System

and Victor3 1420 multilabel plate reader. Luminescence was determined through counts

per second (CPS) output and receptor activity was normalized to vehicle responses for

each condition and reported as % basal activation.

GPCR Receptor trafficking assays:

Wild type and mutagenized receptors were cloned in frame into pcDNA3 CFP or

pcDNA3 YFP vectors and transfected into HEK-293T cells. Following transfection,

~100,000 cells were transferred to a glass cover slip and fixed with 2%

paraformaldehyde. Plasma membranes were then stained with 5mg/mL Wheat Germ

Agglutinin (WGA-594) and imaged on Zeiss 710 LSM confocal microscope. Receptor

localization and trafficking was compared between 5-10 independent cells from three

independent transfections expressing YFP tagged wild-type or mutagenized receptors.

Page 76: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

62

Colocalization of the receptor and plasma membrane was determined through Pearson’s

coefficient, calculated in FIJI software using the coloc2 plugin as previously described16.

Pearson colocalization coefficients of WGA and YFP were obtained for each condition,

and values were Fisher transformed prior to statistical analysis.

RAPTORX Receptor Modeling:

Receptor structures were predicted using full length ORF sequences from wild-type and

mutant leucokinin receptors modeled using the RAPTORX prediction server17 .

Prediction quality was assessed through the computed P value for fit (P<.001), using a

NK1R receptor as a template for prediction, a member of the same receptor subfamily as

the leucokinin receptor. Structural predictions were visualized using PyMol software

suite and the specific tryptophan (WT) or leucine (mutant) was highlighted, as well as N

and C termini.

Page 77: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

63

RESULTS

The WxFG domain is present in disparate receptors from multiple taxa:

The initial description of the WxFG receptor motif focused on the functional roles

of this domain in the human complement C5a receptor9. We sought to systematically

evaluate the prevalence of the WxFG motif among different Class A GPCRs, as the

original description of this motif suggested a high degree of conservation. Subsequently,

we performed a bioinformatic analysis across all 19 Class A receptor subfamilies

focusing on human receptor sequences11. We found that the motif is present in multiple

members of 17 different receptor subfamilies (Table 1). We did find a substitution in two

different peptide receptor subfamilies, A6 and A7, the neuromedin U and cholecystokinin

receptor subtypes respectively, in which a phenylalanine (F) residue has replaced the

tryptophan (W) position. Notably, this motif is completely absent in subfamilies A13 and

A14. Both of these receptor subfamilies have nucleotide/lipid ligands and suggest a

secondary loss of this motif in these related receptor subtypes.

Having established that this motif is present in multiple receptor subtypes, we

next evaluated whether this motif was a common feature of each receptor family across a

number of different taxa. We extended our analysis focusing on receptor sequences from

established vertebrate and invertebrate model organisms: Homo sapiens, Mus musculus,

Gallus gallus, Xenopus laevis, Danio rerio, Ciona intestinalis, Drosophila melanogaster,

and Caenorhabditis elegans. We aligned the sequence corresponding to the extracellular

loop for all clear members of a receptor family for each of taxa. Notably, in 15 of the 17

receptor subfamilies, the W position exhibited the least identity variance across all

Page 78: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

64

sequences as indicated by seq2logo bitscore (Figure 1). As noted previously, the

Cholecystokinin and Neuromedin U receptor subfamilies show a common F substitution.

The W residue is critical for GPCR signaling:

Alteration of the W residue in the WxFG motif causes a loss of receptor signaling

in the human complement 5a factor receptor, C5aR 9. Given the widespread prevalence

of this motif, we next examined whether the functional aspects of this motif were similar.

We first focused on the effects of different amino acid substitutions on receptor signaling

using the leucokinin receptor (LKR) from Drosophila, a receptor with critical roles in

meal size discretion and diuresis 18,19. We made different amino acid substitutions

representing changes to non-polar, charged, and aromatic subclasses: W101A, W101L,

W101K, W101E, and W101F. Each of these receptor variants, with the exception of the

W101F, were insensitive to ligand presentation. Specifically, while wild type LKR

exhibited dose dependent responses to ligand, the W101L, W101K, W101A, and W101E

variants exhibited no significant response to ligand presentation (Figure 2). In contrast,

the W101F substitution showed wild-type responses to ligand presentation, indicating

that this variant encodes a functional leucokinin receptor. Collectively, these data suggest

that amino acid identity at the W position in the WxFG motif is critically important for

receptor function.

We next extended our observations to include receptors from several different

subfamilies, and that differ in their signaling properties. We targeted six additional

receptors representing Class A GPCR subfamilies A4, A5, A6, A10, and A16.

Additionally, these receptors couple to distinct intracellular heterotrimeric G proteins,

with the corazonin receptor (CRZR) and follicle-stimulating hormone receptor (FSHR)

Page 79: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

65

coupling to Gs 20,21, the adipokinetic hormone receptor (AKHR), muscarinic

acetylcholine receptor (mACHR1), and leucokinin receptor (LKR) coupling to Gq 22–24,

and GALR1 and SSTR2 coupling to Gi 25,26. The promiscuous Gα16 subunit was

included in GALR1 and SSTR2 transfections to promote coupling of these receptors to

elevated calcium levels for ease of monitoring. All SSTR2, GALR1, mACHR1, and

FSHR mutant variants exhibited a significant loss of function compared to their wild type

counterparts. Additionally, the Drosophila AKH and corazonin receptors (AKHR,

CRZR), exhibited a similar loss of function when mutagenized (Figure 3).

Tryptophan variants impair constitutive signaling:

As previous studies suggested that WxFG domain mutant receptors bind ligand,

but lack signaling responses9, we tested whether the loss of signaling phenotypes could

be rescued by simultaneously conferring constitutive activity in a mutagenized receptor

background. Many GPCRs exhibit constitutive activity and constitutive activity can be

experimentally induced through targeted mutagenesis of the DRY motif 18,19. To induce

constitutive activity, we mutagenized the aspartate residue in the DRY motif of

the Drosophila AKHR receptor (D136A), which conferred significantly elevated basal

signaling compared to wild type AKHR (Figure 4A), whereas the W105L variant showed

no signaling response (Figure 4B). In contrast, a W105L, D136A double mutant receptor

showed no signaling activity in response to ligand or in the basal state, indicating a loss

of both ligand responsiveness and constitutive activity for that receptor (Figure 4C).

Page 80: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

66

Variants in the W residue impairs receptor trafficking:

Given that substitutions in the WxFG domain cause a reduction in signaling

independent of receptor type and ablate constitutive activity, we reasoned that abnormal

receptor expression could potentially explain the loss of function phenotypes. To

determine the impact of the tryptophan substitution in the WxFG motif on receptor

expression, we incorporated a C-terminal fluorescent tag. We targeted three different

receptors that differ in their signaling properties and are members of different receptor

subfamilies. Specifically, we added a yellow fluorescent protein (YFP) to the Drosophila

CRZR, and LKR and used a previously generated human 5HT2c-YFP receptor in both

wild-type and W�L substitution variants. While we observed strong fluorescent signals

in both wild-type and mutant receptors, however, the patterns of fluorescence were very

different. Specifically, we observed strong YFP signal limited to the plasma membrane

in wild-type receptors. In each of the W�L receptor variants, we found a dramatic

reduction in YFP signal at the plasma membrane, with an increased amount of

intracellular YFP expression (Figure 5). These results suggest that the WxFG motif plays

a critical role in receptor trafficking and provide a mechanism to explain the loss of

function receptor phenotypes.

Structural modeling suggests the W stabilizes receptor architecture:

Based on the confluence of our trafficking and signaling data, we hypothesized

that mutagenesis of the conserved W in WxFG must be significantly altering receptor

conformation and folding. To assess the impact of these substitutions, we initially

modeled the leucokinin receptor and a variant (W101L) using the RAPTORX protein

structural modeling program. The models suggest that substitutions of the tryptophan

Page 81: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

67

residue result in displacement of the N and C terminal regions of the receptor (Figure 6).

This distortion in overall receptor conformation suggests receptor instability may explain

the aberrant trafficking of mutant receptors.

Page 82: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

68

DISCUSSION

In this study, we extended a phylogenetic analysis of the presence of the WxFG

domain and found that this domain is highly conserved throughout different Class A

GPCRs. Specifically, this domain is present in seventeen of the nineteen Class A

subfamilies and is a prominent feature of these receptors independent of taxa. We also

evaluated the functional contributions of the largely invariant tryptophan reside and

found that an aromatic residue in this position is required for receptor function,

independent of receptor type or specific downstream effector. Furthermore, we find that

that this domain functions in GPCR trafficking and propose a structural model in which

the tryptophan stabilizes overall receptor architecture.

In the initial investigation of WxFG motif in the C5a receptor, Klco et al. noted

that a tryptophan was present in the first extracellular loop in 80% of human peptide-

binding GPCRs, and that a phenylalanine was present in 10 % of these receptors,

indicating high conservation of an aromatic residue at this position9. Here we performed a

comprehensive analysis of the presence of the WxFG motif in each Class A GPCR

subfamilies and showed that the motif is largely present in all subfamilies, with the

exception of A13 and A14 subfamilies. The loss of this domain in these subfamilies is

interesting, as it suggests that A13 and A14 receptor subfamilies may utilize different

strategies to adopt stable conformations. This hypothesis is supported by a

multidimensional scaling analysis by Pele et al. which suggests that the WxFG motif co-

evolved with proline residues on TM2 and TM5, presumably stabilizing overall receptor

structure 10. Notably, these proline residues are absent in A13 GPCRs, while the TM5

proline is absent in A14 members, both of which lack the WxFG motif. Additionally, the

Page 83: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

69

motif does not appear to be conserved in any other GPCR subfamily, suggesting it arose

during the expansion of Class A GPCRs27.

Multiple previous studies have investigated the WxFG motif in individual

receptors 9,28–30, and we have furthered these initial descriptions and suggest a novel

mechanism for the role of this motif in GPCR function. The initial investigation of the

C5a receptor showed that mutations at the tryptophan residue generated receptors that

bind ligand but are unable to transduce a signaling response9. Our data suggests that

modified receptors show an aberrant distribution within the cell, meaning that even if

these receptors are able to bind ligand, they are not present at the plasma membrane.

Given that these previous ligand binding studies were performed on isolated membrane

preparations, we suspect that modified receptors are localized to intracellular membrane

compartments. In support of this, A W99C substitution in the NK2R receptor exhibited

no ligand binding when assayed on whole cells31. The totality of these studies suggests an

aromatic amino acid at the W position is critical for wild type receptor function, and

modification of this position causes abnormal GPCR localization.

The aberrant localization of WxFG mutants might stem from defects in receptor

folding. Many conserved GPCR motifs play roles in stabilizing the receptor’s active and

inactive states, such as the ionic lock/DRY motif 32 and NPxxY motif 33, or act as

microswitches gating receptor activation, such as the CWxP motif 10. As previous studies

have shown that WxFG mutants are able to bind ligand, our data therefore suggest that

the WxFG motif plays an unexpected role in appropriate receptor trafficking. It is

presently unclear if this motif is involved in trafficking to the plasma membrane, or

alternatively, has other unexpected receptor phenotypes that could explain its intracellular

Page 84: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

70

distribution, such as constitutive desensitization of the receptor 34. Many GPCRs require

interactions with accessory proteins for appropriate cell surface trafficking, with the

Calcitonin receptors requiring interaction with RAMPs35, while the β1AR is stabilized at

the cell surface by interactions with PSD-95, a PDZ domain containing chaperone36. The

5HT2c receptor possesses a C-terminal PDZ-binding domain which plays a critical role

in the synaptic localization of this receptor37. Disruption of the WxFG motif may

interfere with receptor-chaperone interactions, leading to the aberrant receptor

localization that we observed. It is possible that the WxFG motif acts as a microswitch,

and its modification interferes with the transition between inactive and active receptor

conformations. We consider this unlikely, as we would predict that a constitutively

active, internalized receptor should still show evidence of increased basal activity.

Alternatively, the c-terminal F(X)6LL domain is critical for α2B-AR and AT1R exit from

the ER8, and WxFG may play a similar role in ensuring appropriate receptor localization.

Furthermore, the cysteine residue downstream of WxFG at the top of TM3 forms a

disulfide bond with a cysteine residue on EL238, and this disulfide bond is required for

appropriate trafficking of M3 receptor to the cell surface. Thus, the WxFG motif may be

important for allowing the interactions between these two extracellular loops in

establishing the appropriate receptor topology.

This study represents the most comprehensive investigation of the WxFG motif

across multiple Class A GPCR subfamilies to date. We have shown that this motif is

heavily conserved across Class A GPCRs, and substitution of the W with a nonaromatic

amino acid yields a nonfunctional receptor with impaired plasma membrane localization.

We suggest a novel mechanism by which that the WxFG motif plays a critical role in

Page 85: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

71

wild type GPCR trafficking to the plasma membrane, and likely functions in concert with

other conserved GPCR motifs to stabilize the receptor in an appropriate conformation.

Further investigation is necessary to determine whether these mutant receptors ever reach

the cell surface and are retained in the ER-Golgi complex, or that receptor instability

causes rapid internalization of the receptor.

ACKNOWLEDGEMENTS

We acknowledge Dr. Glen Marrs for microscopy assistance, Dr. Cecil Saunders and Jon

Nelson for manuscript editing, Dr. T. Michael Anderson for statistical analysis, and Dr.

Katherine Herrick-Davis for reagents. This work was funded by NSF IOS1355097 to

ECJ, and the WFU Center for Molecular Signaling (CMCS).

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest with the contents of this

manuscript.

AUTHOR CONTRIBUTIONS

MJR and ECJ designed experiments, MJR, JPE, MB performed experiments. MJR and

ECJ analyzed results. MJR and ECJ wrote manuscript, all authors contributed edits.

Page 86: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

72

REFERENCES

1. Whitehead, I. P., Zohn, I. E. & Der, C. J. Rho GTPase-dependent transformation

by G protein-coupled receptors. Oncogene 20, 1547–1555 (2001).

2. Overington, J. P., Al-Lazikani, B. & Hopkins, A. L. How many drug targets are

there? Nat. Rev. Drug Discov. 5, 993–996 (2006).

3. Lefkowitz, R. J. A Brief History of G-Protein Coupled Receptors (Nobel Lecture).

Angew. Chemie Int. Ed. 52, 6366–6378 (2013).

4. Hanlon, C. D. & Andrew, D. J. Outside-in signaling – a brief review of GPCR

signaling with a focus on the Drosophila GPCR family. J Cell Sci 128, 3533–3542

(2015).

5. Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor.

Science (80-. ). 289, 739–745 (2000).

6. Olivella, M., Caltabiano, G. & Cordomí, A. The role of Cysteine 6.47 in class A

GPCRs. BMC Struct. Biol. 13, 3 (2013).

7. Chen, W. J., Goldstein, J. L. & Brown, M. S. NPXY, a sequence often found in

cytoplasmic tails, is required for coated pit-mediated internalization of the low

density lipoprotein receptor. J. Biol. Chem. 265, 3116–3123 (1990).

8. Duvernay, M. T., Zhou, F. & Wu, G. A Conserved Motif for the Transport of G

Protein-coupled Receptors from the Endoplasmic Reticulum to the Cell Surface. J.

Biol. Chem. 279, 30741–30750 (2004).

9. Klco, J. M., Nikiforovich, G. V & Baranski, T. J. Genetic Analysis of the First and

Page 87: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

73

Third Extracellular Loops of the C5a Receptor Reveals an Essential WXFG Motif

in the First Loop. J. Biol. Chem. 281, 12010–12019 (2006).

10. Pelé, J., Abdi, H., Moreau, M., Thybert, D. & Chabbert, M. Multidimensional

Scaling Reveals the Main Evolutionary Pathways of Class A G-Protein-Coupled

Receptors. PLoS One 6, e19094 (2011).

11. Joost, P. & Methner, A. Phylogenetic analysis of 277 human G-protein-coupled

receptors as a tool for the prediction of orphan receptor ligands. Genome Biol. 3,

RESEARCH0063 (2002).

12. Weiner, M. P. et al. Site-directed mutagenesis of double-stranded DNA by the

polymerase chain reaction. Gene 151, 119–123 (1994).

13. Edelheit, O., Hanukoglu, A. & Hanukoglu, I. Simple and efficient site-directed

mutagenesis using two single-primer reactions in parallel to generate mutants for

protein structure-function studies. BMC Biotechnol. 9, 61 (2009).

14. Johnson, E. C. et al. Identification of Drosophila neuropeptide receptors by G

protein-coupled receptors-beta-arrestin2 interactions. J. Biol. Chem. 278, 52172–8

(2003).

15. Offermanns, S. & Simon, M. I. Gα15 and Gα16 Couple a Wide Variety of

Receptors to Phospholipase C. J. Biol. Chem. 270, 15175–15180 (1995).

16. Irannejad, R. et al. Functional selectivity of GPCR-directed drug action through

location bias. Nat. Chem. Biol. 13, 799–806 (2017).

17. Källberg, M. et al. Template-based protein structure modeling using the RaptorX

Page 88: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

74

web server. Nat. Protoc. 7, 1511–1522 (2012).

18. Al-Anzi, B. et al. The leucokinin pathway and its neurons regulate meal size in

Drosophila. Curr. Biol. 20, 969–978 (2010).

19. Yang, M. Y., Wang, Z., MacPherson, M., Dow, J. A. T. & Kaiser, K. A novel

Drosophila alkaline phosphatase specific to the ellipsoid body of the adult brain

and the lower Malpighian (renal) tubule. Genetics 154, 285–297 (2000).

20. Sha, K. et al. Regulation of ethanol-related behavior and ethanol metabolism by

the corazonin neurons and corazonin receptor in Drosophila melanogaster. PLoS

One 9, (2014).

21. Costagliola, S. et al. Tyrosine sulfation is required for agonist recognition by

glycoprotein hormone receptors. EMBO J. 21, 504–513 (2002).

22. Baumbach, J., Xu, Y., Hehlert, P. & Kühnlein, R. P. Gαq, Gγ1 and Plc21C Control

Drosophila Body Fat Storage. J. Genet. Genomics 41, 283–292 (2014).

23. Biddlecome, G. H., Berstein, G. & Ross, E. M. Regulation of Phospholipase C-1

by G and m1 Muscarinic Cholinergic Receptor. Steady-state balance of receptor-

mediated activation and GTPase-activating protein-promoted deactivation. J. Biol.

Chem. 271bara, 7999–8007 (1996).

24. Terhzaz, S. et al. Isolation and characterization of a leucokinin-like peptide of

Drosophila melanogaster. J. Exp. Biol. 202, 3667–3676 (1999).

25. Kagimoto, S. et al. Human somatostatin receptor, SSTR2, is coupled to adenylyl

cyclase in the presence of Gi alpha 1 protein. Biochem. Biophys. Res. Commun.

Page 89: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

75

202, 1188–1195 (1994).

26. Habert-Ortoli, E., Amiranoff, B., Loquet, I., Laburthe, M. & Mayaux, J. F.

Molecular cloning of a functional human galanin receptor. Proc. Natl. Acad. Sci.

U. S. A. 91, 9780–9783 (1994).

27. Nordström, K. J. V, Sällman Almén, M., Edstam, M. M., Fredriksson, R. &

Schiöth, H. B. Independent HHsearch, Needleman–Wunsch-Based, and Motif

Analyses Reveal the Overall Hierarchy for Most of the G Protein-Coupled

Receptor Families. Mol. Biol. Evol. 28, 2471–2480 (2011).

28. Peeters, M. C. et al. GPCR structure and activation: an essential role for the first

extracellular loop in activating the adenosine A2B receptor. FASEB J. 25, 632–643

(2010).

29. Ragnarsson, L., Andersson, Å., Thomas, W. G. & Lewis, R. J. Extracellular

Surface Residues of the α1B-Adrenoceptor Critical for G Protein–Coupled

Receptor Function. Mol. Pharmacol. 87, 121–129 (2015).

30. Ragnarsson, L. et al. Conopeptide ρ-TIA Defines a New Allosteric Site on the

Extracellular Surface of the α1B-Adrenoceptor. J. Biol. Chem. 288, 1814–1827

(2013).

31. Labrou, N. E., Bhogal, N., Hurrell, C. R. & Findlay, J. B. C. Interaction of Met297

in the Seventh Transmembrane Segment of the Tachykinin NK2 Receptor with

Neurokinin A. J. Biol. Chem. 276, 37944–37949 (2001).

32. Audet, M. & Bouvier, M. Restructuring G-Protein- Coupled Receptor Activation.

Page 90: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

76

Cell 151, 14–23 (2012).

33. Fritze, O. et al. Role of the conserved NPxxY(x)5,6F motif in the rhodopsin

ground state and during activation. Proc. Natl. Acad. Sci. U. S. A. 100, 2290–2295

(2003).

34. Barak, L. S., Oakley, R. H., Laporte, S. A. & Caron, M. G. Constitutive arrestin-

mediated desensitization of a human vasopressin receptor mutant associated with

nephrogenic diabetes insipidus. Proc. Natl. Acad. Sci. U. S. A. 98, 93–98 (2001).

35. McLatchie, L. M. et al. RAMPs regulate the transport and ligand specificity of the

calcitonin-receptor-like receptor. Nature 393, 333–339 (1998).

36. Dunn, H. A. & Ferguson, S. S. G. PDZ Protein Regulation of G Protein–Coupled

Receptor Trafficking and Signaling Pathways. Mol. Pharmacol. 88, 624–639

(2015).

37. Bécamel, C. et al. Synaptic multiprotein complexes associated with 5‐HT2C

receptors: a proteomic approach. EMBO J. 21, 2332–2342 (2002).

38. Zeng, F.-Y., Soldner, A., Schöneberg, T. & Wess, J. Conserved Extracellular

Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper

Receptor Cell Surface Localization but Not for G Protein Coupling. J. Neurochem.

72, 2404–2414 (1999).

Page 91: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

77

Sub-

family Subfamily Subtype Human

Gene ECL 1 sequence UniProt entry

A1 Chemokine CCR1 DYKLKDDWVFGDAMCK P32246[92-107]

A2 Chemokine CXCR3 VDAAVQWVFGSGLCK P49682[111-125]

A3 Angiotensin AGTR1 TAMEYRWPFGNYLCK P30556[88-102]

Bradykinin B1 NQFNWPFGALLCR P46663[99-111]

A4 Somatostatin SSTR1 RHWPFGALLCR P30872[121-131]

Opioid OPRD1 METWPFGELLCK P41143[111-122]

A5 Galanin GALR1 QATVYALPTWVLGAFICK P47211[92-109]

A6 Cholecystokinin** CCK KDFIFGSAVCK P32238[105-115]

Neuropeptide FF NPFFR1 VDNLITGWPFDNATCK Q9GZQ6[102-117]

GnRH GNRHR DGMWNITVQWYAGELLCK P30968[98-115]

Orexin HCRTR1 SLLVDITESWLFGHALC O43613[103-119]

A7 Bombesin BRS3 DATHYLAEGWLFGRIGCK P32247[104-121]

TRH* TRHR TDSIYGSWVYGYVGCL P34981[84-99]

Neuromedin U* NMUR1 YEMWHNYPFLLGVGGCYFRT Q9HB89[119-138]

A8 Formyl peptide receptor fMLPR RKAMGGHWPFGWFLCKF P21462[84-100]

Anaphylatoxin C3A HLALQGQWPYGRFLCK Q16581[81-96]

A9 Melatonin MTNR1A LMSIFNNGWNLGYLHCQV P48039[85-102]

Tachykinin TAC1R VVNFTYAVHNEWYYGLFYCK P25103[87-106]

NPY NPY1R FVYTLMDHWVFGEAMCKLN P25929[98-116]

A10 FSH FSHR DIHTKSQYHNYAIDWQTGAGCD P23945[422-443]

A11 Purinergic P2Y1R YYFNKTDWIFGDAMCKL P47900[110-126]

Free Fatty Acid FFAR2 PFKIIEAASNFRWYLPKVVCAL O15552[63-84]

A12 P2 purinoreceptor* P2RY13 KILSDSHLAPWQLRAFVCR Q9BPV8[99-117]

A13 Cannaboid** CNR2 NFHVFHGVDSKA P34972[93-104]

Lysophospatidic acid LPAR1 NTRRLTVSTWLLRQ Q92633[112-125]

Syphingophospate* S1PR2 VTLRLTPVQWFARE O95136[96-109]

Melanocortin** MC1R ETAVILLLEAGALVARAAVLQQLD Q01726[94-118]

A14 Prostoglandins* PTGER3 VYLSKQRWEHIDPSGRLCT P43115[113-131]

A15 Proteases* F2RL1 KIAYHIHGNNWIYGEALCN P55085[131-149]

A16 Opsins* OPN4 TSSLYKQWLFGETGCE Q9UHM6[129-144]

A17 Serotonin HTR2A LTILYGYRWPLPSKLC P28223[133-148]

Dopamine DRD1 GFWPFGSFC P21728[88-96]

Adrenergic ADRA1A LGYWAFGRVFC P35348[89-99]

Trace Amine TAAR1 MVRSAEHCWYFGEVFCKI Q96RJ0[81-98]

A18 Histamine HRH1 NILYLLMSKWSLGRPLCL P35367[84-101]

Adenosine** ADORA1 NIGPQTYFHTC P30542[70-80]

Muscarinic ACh CHRM1 TTYLLMGHWALGTLACD P11229[83-99]

A19 Serotonin HTR1A LNKWTLGQVTCD P08908[99-110]

Page 92: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

78

Table 1: Comparison of representative extracellular loop 1 sequences across Class A

GPCR subfamilies. Human GPCR sequences were obtained from NCBI databases and

extracellular loop 1 sequences were predicted using the TMHMM server. The presence of

the WxFG motif tryptophan residue is highlighted in red, whereas a phenylalanine

residue at this position is highlighted in yellow. A tryptophan is present at the

appropriate position in 15 of the 19 subfamilies, and a tryptophan or phenylalanine is

present in 17 of the 19 subfamilies, indicating a high level of conservation of this motif

amongst class A GPCRs.

Page 93: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

79

Figure 1: Sequence weighting analysis shows that the WxFG motif’s tryptophan

residue exhibits high conservation in Class A GPCR receptor subfamilies.

Extracellular loop 1 sequences from Homo sapiens, Drosophila melanogaster, Danio

rerio, Mus musculus, Caenorhabditis elegans, Xenopus laevis, and Gallus gallus were

obtained using the NCBI database and TMHMM web server, and positional weight

scores were generated using the Seq2Logo application. Heavy conservation of the

tryptophan residue in the WxFG motif is seen in all but family A6, which exhibits greater

conservation of a phenylalanine residue at that position.

Page 94: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

80

Figure 2: Mutagenesis of conserved tryptophan residue in LKR ECL1 ablates

receptor signaling. A. W101L substitution in the WXFG motif of the leucokinin receptor

ablates receptor function. Site-directed mutagenesis of the conserved tryptophan residue

in EL1 yields a receptor which is unresponsive to leucokinin stimulation at all

concentrations. The wild type LKR exhibited a maximal response at 10-6 M ligand

application. B. Substitution of the conserved tryptophan residue in EL1 of the leucokinin

receptor leads to a loss of function when an aromatic residue is not present at that

position. 5 mutagenized variants of LKR were generated and tested using a SRE-luc

signaling assay. Receptor responses were quantified as % basal activity following 10-6 M

ligand presentation. Both WT LKR and the W101F variant exhibited significantly

elevated activity following ligand addition, while variants W101E, W101A, W101K, and

Page 95: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

81

W101L exhibited no significant increase in signaling from baseline (P<.003). W101F

exhibited no significant difference in signaling response when compared to wild type.

Page 96: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

82

Figure 3: Leucine substitution for the conserved tryptophan residue in extracellular

loop 1 leads to a loss of function in multiple receptor types. Receptor constructs were

obtained from multiple repositories and mutagenized as previously described. These

receptor constructs couple to Gs (AKHR, FSHR, white bars), Gq (CRZR, mACHR1,

black bars), or Gi (SSTR2, GALR1, black bars), and the promiscuous Gα16 subunit was

included in transfections of Gi coupled receptors. In each case, leucine substitution at the

W position in the WxFG motif led to a loss of signaling response to 10-6 M ligand

presentation (P<.003), suggesting a conservation of WxFG domain function across taxa.

Specifically, SSTR2, GALR1, FSHR, mACHR1, AKHR, and CRZR wild type receptors

Page 97: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

83

exhibited 246.42%, 210.90%, 764.85%, 231.26%, 845.72%, and 290.66% of basal signal

at 10-6M ligand application, while their respective mutants exhibited a near complete

loss of function (SSTR2 mutant:94.73%, GALR1 mutant:127.29%, FSHR

mutant:123.21%, mACHR1 mutant:138.17%, AKHR mutant: 115.75%, and CRZR

mutant: 104.40% respectively, P<.003, hatched bars).

Page 98: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

84

Figure 4: Substitution of the conserved tryptophan residue to leucine ablates

constitutive activity in a constitutively active AKHR mutant. A. A constitutively

active AKHR receptor was generated by mutagenizing the conserved aspartate residue in

the DRY motif to alanine (D136A). D136A exhibits significantly greater activity at

baseline than the wild-type AKHR receptor, while still remaining responsive to ligand

presentation. B. The W105L substitution eliminates AKHR signaling response following

10-6M ligand presentation. C. The W105L substitution, when incorporated into the

D136A variant, ablated both ligand responsiveness and constitutive activity associated

with the receptor.

Page 99: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

85

Page 100: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

86

Figure 5: The WxFG is critical for proper receptor trafficking. YFP tagged

leucokinin, corazonin, and 5HT2c receptors were mutagenized as previously described

(W�L). Receptor localization was compared between wild type and mutant receptor

variants, using the plasma membrane marker WGA-594 colocalization with YFP signal

to approximate receptor expression at the cell surface. In each case, receptor localization

within the cell was dramatically altered in mutant receptor variants. All wild type

receptors exhibited high levels of colocalization with WGA-594, an expected result given

their function as cell-surface receptors, while each mutant exhibited no significant

colocalization with WGA-594. Taken together, these results suggest impaired receptor

trafficking in WxxL mutants, regardless of receptor background. Wild type 5HT2c

(Fig.5 A-C), (R=.66±.08), LKR (Fig.5 G-I), (R=.78±.07), and CRZR (Fig.5 M-O),

Page 101: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

87

(R=.72±.04), and exhibited strong colocalization with the plasma membrane (black bars),

while their corresponding mutants, 5HT2c W120L (Fig. 5 E-F), (R= -.02±.05), LKR

W101L (Fig. 5 J-L), (R=.05±.10), and CRZR W191L (Fig. 5 P-R), (R= -.11±.12), and

exhibited a dramatic reduction in plasma membrane localization (white bars), with

P<.001 for all wild type – mutant comparisons (Fig. 5 S).

Page 102: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

88

Figure 6: Putative tertiary structures of wild type LKR and mutant W101L are

superimposed to identify gross changes in receptor topology. A. Mutant structures are

modeled in red, and wild type structures are modeled in blue. The superimposed

structures display perfect alignment of the TMs, but a significant distortion of the N-

terminus when a leucine residue is added in place of W101 (B). Thus, LKR may not be

able to tolerate substitutions at W101 due to tryptophan’s stabilization of ECL1 geometry

in relation to the N-terminus and adjacent TMs.

Page 103: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

89

CHAPTER III: Homodimerization of Drosophila Class A Neuropeptide GPCRs:

Evidence for conservation of GPCR dimerization throughout metazoan evolution.

Michael J. Rizzo, Erik C. Johnson

The work in this chapter will be submitted to the journal Biochemical and

Biophysical Research Communications. Experiments were conceived by MJ Rizzo and

EC Johnson. Reagents were generated and experiments were performed by MJ Rizzo.

Data were analyzed by MJ Rizzo and EC Johnson. The manuscript was drafted by MJ

Rizzo and edited by EC Johnson. This study was funded by NSF and WFU CMS.

Page 104: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

90

ABSTRACT

While many instances of GPCR dimerization have been reported for vertebrate

receptors, GPCR dimerization amongst invertebrates remains poorly investigated, with

few invertebrate GPCRs having been shown to assemble as dimers. To date, no

Drosophila GPCRs have been shown to assemble as dimers. Furthermore, dimerization

studies are largely confined to vertebrate organisms, and the extent of GPCR

dimerization amongst invertebrates remains largely overlooked. To explore the

evolutionary conservation of GPCR dimerization, we employed an acceptor-

photobleaching FRET methodology to evaluate whether multiple subclasses of

Drosophila GPCRs assembled as homodimers when heterologously expressed in HEK-

293T cells. We C-terminally tagged multiple Drosophila neuropeptide GPCRs that

exhibited structural homology with a vertebrate GPCR family member previously shown

to assemble as a dimer with CFP and YFP fluorophores and visualized these receptors

through confocal microscopy. FRET responses were determined based on the increase in

CFP emission intensity following YFP photobleaching for each receptor pair tested. For

each receptor expressed as a homodimer pair, a significant FRET response was seen,

while non-significant FRET responses were displayed by both cytosolic CFP and YFP

expressed alone, and a heterodimeric pair of receptors from unrelated families,

suggesting that receptors exhibiting positive FRET responses assemble as homodimers at

the plasma membrane. These results are the first to suggest that Drosophila GPCRs

assemble as homodimeric complexes, and suggest that GPCR dimerization arose early in

metazoan evolution and likely plays an important and underappreciated role in the

cellular signaling of all animals.

Page 105: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

91

INTRODUCTION

G-protein coupled receptors (GPCRs) are the largest superfamily of metazoan cell

surface receptors and are responsible for transducing a wide range of extracellular stimuli

into cellular responses1. These receptors possess a characteristic seven-transmembrane

architecture, with an extracellular N terminus and intracellular C terminus. GPCRs are

essential for a variety of behaviors and physiologies, including vision, taste, homeostatic

regulation, and reproduction2. Given their diverse roles in physiology and behavior, it is

unsurprising that GPCRs are the molecular targets of approximately 50% of

pharmaceuticals3. As a consequence of GPCRs importance in a diverse set of

physiologies and behaviors and their constituting the pharmacological targets of many

drugs, specific determination of their mechanism of actions and signaling pathways are

active areas of research and of widespread biological interest. One phenomenon that has

engendered significant interest is determination of the exact molecular organization of

GPCRs. The first evidence of higher order GPCR structures stemmed from the

identification that functional GABAB receptors consist of two distinct subunits.

Specifically, the GABABR1 and GABABR2 subunits are required to construct the

functional receptor, as the latter subunit is required for trafficking of the GABABR1

subunit to the plasma membrane4. The necessity of dimerization for receptor function is

now recognized as a hallmark of Class C GPCRs5.

Subsequent to the discovery of GABA receptor dimerization, many other

unrelated GPCR dimers have been identified, with a diverse array of phenotypes

attributable to this molecular organization6. While there is clear evidence that GPCRs

can assemble as dimers, the prevalence of GPCR dimerization as it pertains to phylogeny

Page 106: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

92

as well as receptor type remains unresolved. It is clear that GPCR dimerization frequency

varies by receptor class. Class C GPCRs, which include GABAB and metabotropic

glutamate receptors, function as obligatory dimers as previously discussed5. However,

the question of the whether other GPCR subtypes assemble as dimers is the subject of

much debate. Multiple Class A and Class B GPCRs have been shown to assemble as

homo- and/or heterodimers. Dimerization is especially common amongst biogenic amine

receptors, as all 5HT receptors and multiple dopamine receptors have been

experimentally shown to assemble as homo- or heterodimers at the plasma membrane7–9.

Dimerization has also been demonstrated in a number of peptide receptor family

members, including somatostatin receptors, bradykinin receptors, and multiple opioid

receptors, among others, further suggesting oligomeric assembly may be a common

feature of GPCR biology10–12. A difficulty in determining the extent to which Class A and

B form dimers is that these receptors structures may not be as stable as the Class C

GPCRs, and in fact these higher order structures may be transient or dynamically

regulated for receptors in these classes, which thus reduces the probability of finding

GPCR oligomers13. Thus, the possibility exists that the pool of GPCRs at the cell surface

may in fact be a heterogeneous mixture of monomeric, dimeric and higher order

oligomeric structures 13–15.

One example of dimerization imparting differential receptor function are the

gonadotropin releasing hormone (GnRH) receptors in Ciona intestinalis, where variance

in receptor dimerization influences intracellular cAMP and Ca2+ signaling 16. Specifically,

Ciona expresses four specific variants of GnRH receptors, GnRHR1-GnRHR4, which

have been shown to form both homo-and heterodimers when these receptors are

Page 107: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

93

coexpressed. Intriguingly, GnRHR1 homodimers elicit a ten-fold reduced response

compared to the heterodimer, but homodimers of GnRHR1 were able to signal through

both cAMP and Ca2+. Conversely, the GNRHR1-4 heterodimer signaled through only

Ca2+, suggesting that receptor dimerization is responsible for fine tuning cellular

responses to GnRH signaling in this species.

Furthermore, a growing body of evidence supports the notion that a dimeric

GPCR functions as the fundamental signaling unit for many receptors17. Perhaps the best

example of this phenomenon stems from investigations of the human 5HT2c receptor

homodimer. Multiple studies have confirmed that the 5HT2c receptor assembles as a

homodimer, and that this dimerization occurs prior to the mature receptor expression at

the plasma membrane, suggesting that dimerization may be necessary for appropriate

receptor trafficking to the cell surface18–20. To determine whether a homodimer

represented the functional receptor molecule for this receptor, Herrick-Davis et al.

generated a ligand-binding and signaling deficient mutant 5HT2c resulting from a single

amino acid substitution (S138R)21. They then co-expressed these mutant receptors with

wild type 5HT2c receptors in HEK293 cells. Remarkably, the group found that not only

did the mutant S138R 5HT2c variant retain its ability to homodimerize with wild-type

5HT2c receptors (as resolved by both FRET and Co-IP interactions), but additionally that

5HT signaling through this receptor complex was significantly impaired when these

receptors were co-expressed when compared to cells expressing wild type copies of the

5HT2c receptor alone. These experiments provide perhaps the strongest evidence that, at

least for some Class A GPCRs, homodimerization is fundamental to their ability to bind

ligands and transduce this event into appropriate cellular responses.

Page 108: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

94

Studies of GPCR dimerization have largely been limited to investigations of

vertebrate receptors, notably those from humans, rats, and mice7. In contrast, there is a

dearth of reports on invertebrate GPCR dimerization, and little is known as to the

function and frequency of GPCR dimerization amongst invertebrates. The previously

discussed study on Ciona intestinalis demonstrated that dimerization amongst Class A

GPCRs (GnRH receptors) not only occurred, but acted as a significant modulator of

GnRH signaling in the ascidian16. Still, no further studies on Ciona have indicated any

additional species-specific GPCRs in dimerization. Additionally, studies on

Caenorhabditis elegans, despite an estimated 1000 GPCRs present in their genome, have

revealed only a single putative GPCR dimer pair – a heterodimer between the receptors

DAF-37 and DAF-3822. This dimer also plays a significant functional role in the

organism, as it is necessary for proper dauer formation during C. elegans development.

Drosophila melanogaster, another popular invertebrate model organism, has yet to have

GPCR dimerization demonstrated amongst any receptors present in its genome, although

it should be noted that Drosophila GABAB receptors, similar to their mammalian

counterparts, require co-expression of R1 and R2 subunits to confer proper GABA

responsiveness23. While this finding is consistent with heterodimerization between these

two receptors, no FRET, Co-IP, or other dimerization-specific methodology was

employed to verify that such an interaction did in fact occur. Overall, the lack of

investigation of GPCR dimerization in invertebrates obfuscates our understanding of the

function of these receptors in their respective organisms, and also hinders efforts to

explore the evolution of GPCR dimerization with appropriate rigor.

Page 109: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

95

As previously noted, to date, no Drosophila GPCR’s have been demonstrated to

assemble as dimers. In this study, rather than focus on a singular receptor, as is a

common approach in the extant literature, we chose to adopt a systematic approach

informed by evolutionary homology to other GPCRs previously shown to dimerize in

other taxa in order to identify whether Class A (Rhodopsin-like) Drosophila GPCRs,

specifically those involved in neuropeptide signaling, assembled as dimers. We chose to

evaluate seven different Drosophila neuropeptide GPCRs that belong to six Class A

receptor subfamilies that have been previously been shown to assemble as dimers in other

species, to determine whether these Drosophila receptors formed higher order structural

ensembles at the plasma membrane. To this aim, we employed a Fluorescence Resonant

Energy Transfer (FRET) based approach, as this is a standard assay to investigate

intermolecular interactions. Specifically, FRET assays rely on the transfer of energy

from one fluorescent donor to an acceptor molecule and are predicated on short

intermolecular distances between the two fluorophores24.

In this study, prospective dimeric pairs of both CFP and YFP tagged Drosophila

GPCRs were transiently expressed in HEK-293T cells and assessed for FRET responses.

Significant FRET efficiencies were observed for each receptor homodimer pair when

compared to controls, as well as cells expressing donor and acceptor receptors from

unrelated receptor families, suggesting that the receptors studied assemble as

homodimers at the cell surface. These results are the first evidence for GPCR

dimerization amongst Class A Drosophila neuropeptide receptors, and the prevalence of

homodimerization across multiple receptor subtypes suggests that GPCR dimerization

Page 110: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

96

has been conserved throughout metazoan evolution and is a feature of the receptor

superfamily.

Page 111: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

97

METHODS

GPCR cloning and fusion protein generation.

All GPCRs used in this study were previously subcloned into pcDNA3 or

pcDNA5 expression vectors (Table 1), with the exception of the 5HT2c-CFP and 5HT2c-

YFP pair, which were generously donated by Dr. Katharine Herrick-Davis. PCR primers

were designed to add N and C terminal restriction sites to facilitate directional cloning

into the pcDNA3 CFP and pcDNA3 YFP expression vectors (Table 2). Likewise, PCR

products were designed to eliminate the stop codon and make sure that the resulting

receptor reading frame would be continuous with the CFP or YFP reading frames. All

resulting plasmids were sequence verified and plasma membrane expression was verified

for each receptor prior to FRET analysis.

Cell culture and transfection

HEK-293T cells were grown in a standard growth medium of Dulbecco’s

modified Eagle medium (DMEM) supplemented with fetal bovine serum,

antibiotic/antimycotic, and 2mM L-glutamine. Prior to transfection, cells were split and

seeded into 24 well dishes. Transfections were performed using Lipofectamine 2000

transfection reagent in serum free Opti-MEM media when cell density reached ~0.2*106

cells/mL in each well. For all co-expression experiments, receptor cDNAs were

transfected at a 1:2 CFP/YFP ratio to bias CFP tagged receptors to dimerize with YFP

tagged receptors. Transfected cells were split into glass bottom dishes and allowed to

recover for 24 hours in standard growth media following transfection, at which point

Page 112: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

98

media was switched to clear, modified Eagle medium (MEM) for imaging analysis. All

imaging experiments were performed 48 hours following transfection.

Signaling assays

To assess receptor function in tagged receptor variants, we performed signaling

assays on two of the seven modified receptors. YFP tagged variants of the LKR and

CRZR receptors, as well as wild type variants for each receptor for each receptor25, were

transfected along with the SRE-luciferase reporter construct at a 5:1 ratio into HEK-293T

cells using Lipofectamine 2000. Following transfection, cells were split into 96 well

plates and given an additional 24 hours to recover in standard growth media. Following

recovery, media was replaced with clear MEM containing either 10-6M ligand or vehicle

for each condition and left to incubate for four hours. Luciferase activity was assessed

using the SteadyLite Plus Reporter Gene Assay System according to manufacturer’s

protocol and luminescence levels were measured using a Victor3 1420 multilabel plate

reader.

Microscopy and FRET Imaging analysis

For each condition tested, between 4 and 30 cells were visualized using a Zeiss

710 scanning confocal microscope and images were subsequently analyzed using Zen

software. All imaging was performed under identical conditions for quantification

purposes and to facilitate statistical analysis across conditions. Initial fluorescent levels

were determined to gauge CFP and YFP-tagged receptor expression. These values were

used to determine donor and acceptor intensities that were used for subsequent analysis.

Prior to evaluating FRET efficiencies, we imaged cells expressing only cytosolically

Page 113: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

99

expressed CFP and YFP to generate a characteristic emission spectrum for each

fluorophore to be used for linear unmixing analysis. Following an initial imaging

protocol, acceptor photobleaching experiments were performed by defining an area of

interest (AOI) around a region of the cell membrane and applying high intensity, 514nm

laser pulses to photobleach YFP (Figure 1). Time-lapsed images underwent image

analysis to measure the intensity of both CFP and YFP emission both pre and post

photobleaching. Any image where CFP or YFP intensities fell below the intensity of the

residuals channel following linear unmixing was not analyzed. FRET efficiency was

subsequently determined based on the increase of CFP emission intensity following YFP

photobleaching using the formula: FE%=(Dpost-Dpre)/Dpost. FRET efficiencies were

compared across conditions using a one-way ANOVA and a Tukey post-hoc test.

Page 114: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

100

RESULTS

Drosophila receptors were chosen for analysis based on sequence homology to

mammalian receptors previously shown to form dimers (Table 1). We chose seven

receptors that are members of six different Class A receptor subfamilies and that signal

through disparate mechanisms and participate in a diverse set of behaviors and

physiologies. Specifically, the Drosophila corazonin receptor (CRZR) is a member of

the Gonadotropin Releasing Hormone (GnRH) receptor subfamily and is involved in

mediating multiple behaviors in insects, including cardioactivity, gregarious

pigmentation, circadian rhythms, and stress 26–28. GnRH receptors have previously been

shown to dimerize in multiple organisms, including rats, wallabies, and the tunicate

Ciona intestinalis16,29,30. In mammals, neurokinin receptors fulfill an array of functions

ranging from pain perception to vasodilation, and have been show to assemble as dimers,

and we chose to examine two Drosophila receptors related to mammalian neurokinin

receptors, the leucokinin receptor (LKR) and tachykinin receptor at 86C (TAKR86C)31,32.

In mammals, the NPY receptor regulates feeding behaviors, and has been shown to form

higher order structures, thus we chose to evaluate the Drosophila NPF receptor (NPFR),

which like its mammalian homolog impacts feeding behaviors in the fly, for dimer

formation analysis33,34. The proctolin receptor (ProcR) is a member of the thyrotropin-

releasing hormone (TRH) receptor superfamily, whose hormone serves as a master

regulator for pituitary hormone release, and was chosen for analysis as human TRH

receptors receptor have been previously shown to dimerize in heterologous expression

systems33,35,36. We also evaluated the Drosophila allatostatin C receptor 2 (AstC-R2), as

it is a member of the somatostatin family and, in the rat, somatostatin receptors been

Page 115: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

101

shown to form both homo- and heterodimers, with the receptor subclass functioning in

multiple physiologies ranging from sleep regulation to regulation of motor activity 11.

Lastly, we evaluated dimerization in the Drosophila pyrokinin receptor 1 (PK1R), which

is a neuromedin U receptor family member, another receptor family shown to assemble

as homodimers in humans that also functions in a diverse array of physiological

responses, including but not limited to blood pressure regulation, feeding behavior, and

immune system function11,37,38.

Following fluorophore-tagging these receptors, we sought to determine whether

the fluorescent tag interfered with receptor function. We used two parameters to assess

receptor function, the first was an evaluation of plasma membrane expression of the

tagged receptors. First, we only analyzed receptors that showed high levels of expression

at the plasma membrane. Additionally, we evaluated receptor signaling from the LKR-

YFP and CRZR-CFP tagged variants. In both cases, a robust signaling response was

observed at 10-6M ligand concentrations for each tagged receptor that was not

significantly different from the signaling responses exhibited by their respective wild

type receptors (Figure S1). Collectively, these results indicate that fluorophore addition

does not interfere with receptor function for these receptors.

Next, we tested the FRET signatures from cells transfected with both CFP and

YFP tagged receptors. To establish a baseline for non-FRET, we introduced a

cytoplasmic CFP and YFP construct and subjected those cells to the acceptor

photobleaching protocol (Fig. 1). There were minimal FRET signatures observed and we

interpret these as random interactions coincident with localized expression of both

fluorophores. We then compared FRET efficiencies between the 5HT2c receptor and the

Page 116: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

102

cytoplasmic introduction of both fluorophores. The 5HT2c receptor has been shown to

dimerize extensively and serves as a hallmark for the phenomena18–21. Specifically,

cytoplasmic CFP and YFP exhibited a negligible average FRET efficiency of

4.92%±1.28 (Fig. 2 A-F, Fig. 3Q), while the 5HT2c CFP/YFP pair exhibited a statistically

significant higher average FRET efficiency of 15.65%±1.72 (Fig. 2G-L, Fig. 3Q) (P

<0.05 ANOVA). These results indicate that our experimental system is able to accurately

identify bona fide receptor dimers.

Next, we tested the seven Drosophila Class A GPCRs previously described under

the same experimental conditions. Each receptor tested showed FRET efficiencies

significantly different from CFP and YFP alone, but not significantly different from the

5HT2c positive control (Fig. 3). Specifically, the corazonin receptor (CRZR) exhibited the

highest FRET efficiency of all receptors tested at 21.77%±3.07. This was followed by

AstC-R2, which displayed a 21.38%±2.49 FRET efficiency. The NPF receptor, the lone

Gαi-coupled receptor tested in this study, showed a FRET efficiency of 15.77%±0.80.

The two Drosophila tachykinin receptors tested, LKR and TKR86C, showed robust

FRET efficiencies of 10.52%±1.57 and 12.20%±1.85, respectively. Finally, the proctolin

and pyrokinin 1 receptors exhibited the lowest FRET responses of all homodimers tested,

with FRET efficiencies of 9.96%±1.75 and 7.79%±1.30, respectively, although it is

important to note that both receptors FRET responses were significantly higher than

cytosolically expressed CFP and YFP (P<.05), and thus represent strong evidence for

homodimerization amongst these receptors. In contrast, when we introduced a heterotypic

pair of NPFR-CFP and TKR86C-YFP constructs, we observed a FRET response of

5.80%±1.81 that was not significantly different than the CFP/YFP negative control. This

Page 117: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

103

finding is significant as it rules out the alternative interpretation that FRET responses are

a simply a consequence of coexpression of fluorophore-tagged receptors at the plasma

membrane.

Page 118: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

104

DISCUSSION

The results of this investigation provide the first experimental evidence that

multiple Drosophila Class A GPCRs assemble as homodimers at the plasma membrane.

Rather than focus on a singular receptor, we undertook a systematic approach informed

by evolutionary homology to identify seven candidate neuropeptide receptors from six

GPCR families. Notably, for each receptor studied, significant FRET responses

consistent with homodimerization were detected. This suggests that GPCR dimerization

both occurs in Drosophila and is itself a conserved feature of specific GPCR families and

that has been conserved throughout metazoan evolution. Additionally, this investigation

adds to the relatively understudied literature regarding invertebrate GPCR dimerization,

as dimerization had been previously observed only in tunicates and C. elegans, and has

now been demonstrated in dipterans as well.

The receptors examined in this study fulfill diverse physiological roles within the

organism. The corazonin receptor, a member of the GnRH receptor subfamily, is critical

to Drosophila response to myriad stressors, including starvation and desiccation, while

also fulfilling a major role in ethanol metabolism26,27. The tachykinin-related receptors,

TKR86C and LKR, have been implicated in a range of functional roles including

regulation of meal size, sexual activity and fecundity, and the integration of metabolic

state and sleep39–41. The somatostatin family member AstC-R2, while named for its

inhibitory effects on juvenile hormone secretion from the corpora allata, is a key

regulator of multiple physiologies, ranging from circadian rhythm regulation to

nociception and innate immunity, while also serving as a cardioinhibitory peptide in the

fly42–44. The NPF receptor has been shown to mediate physiologies ranging from feeding

Page 119: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

105

and foraging behavior to alcohol sensitivity as well as sleep-wake behaviors45–48. The

proctolin receptor serves as a key regulator of cardioactivity, and has also been shown to

regulate both locomotor activity and thermal preference in Drosophila larvae49,50. Finally,

the pyrokinin-1 receptor, a member of the neuromedin U (NMU) superfamily, has been

implicated in both pheromone biosynthesis as well as the suppression of insulin

production in Drosophila51,52. The diversity of functional roles these receptors fulfill,

coupled with the knowledge that these receptors assemble as higher order structures at

the plasma membrane, suggest that further investigation of the oligomeric assembly of

these receptors within the fly may be critical to dissecting discrete functional roles for

each of the receptors studied.

While there was a significant range of FRET efficiencies reported across the

receptors tested, it is important to consider that FRET efficiency is dependent on a

number of factors beyond whether the two molecules assemble as a dimer, including the

length and orientation of each receptor’s C-terminus (which impacts the distance between

fluorophores), variations in donor/acceptor ratios, and membrane curvature 24,53. As such,

absolute comparisons of FRET efficiencies across receptors do not necessarily reflect

differences in dimerization frequency or the percentage of receptors which assemble as

dimers at the cell surface. Still, this only increases our confidence in our results, as our

measurements of FRET efficiencies for many of the homodimeric pairs investigated were

larger than the 5HT2c receptor pair, and in all cases were significantly higher than

cytosolically expressed CFP and YFP, suggesting that indeed dimerization appears to be

a common phenomenon that is widespread throughout both Drosophila GPCRs and

likely the GPCR superfamily itself, and is not specific to a particular taxon.

Page 120: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

106

We limited our investigation of receptor dimerization only at the plasma

membrane. Therefore, we cannot conclude that dimerization is specific to this cellular

compartment or whether these receptor interactions are exhibited elsewhere in the cell.

Some studies suggest dimerization occurs co-translationally and can be observed in both

the ER and Golgi, as has been shown with the 5HT2c receptor utilized in this study18.

However, other reports suggest that dimerization, especially amongst class A GPCRs, are

transient phenomena and therefore may assemble spontaneously at the plasma membrane,

leading to a dynamic population of monomeric and higher order oligomeric states13. It

would be interesting to see if similar FRET efficiencies for each homodimer pair studied

could be recorded from CFP and YFP tagged receptors as they move through the ER and

Golgi, which would shed light on the biogenesis of Drosophila GPCR dimers.

One aspect of dimerization that was not tested in this study was the impact of

ligand introduction on FRET efficiency between homodimeric receptor pairs. Previous

studies have shown that ligand binding can lead to increased, decreased, or unchanged

FRET responses. This, likely, is the result of the conformational changes which take

place in the receptor molecule following ligand binding and receptor activation54–56.

Future studies should investigate whether ligand introduction alters FRET response

through either promoting or inhibiting dimerization in each of the receptors tested.

Taken together, the results of this study suggest that homodimerization of

Drosophila Class A neuropeptide GPCRs may represent a common feature of G protein-

coupled receptors. This study represents the first step towards a comprehensive analysis

of homodimerization amongst all Drosophila class A GPCRs. It is important to note that

these receptors still represent a fraction of the total neuropeptide receptor GPCRs present

Page 121: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

107

in the Drosophila genome, which contains 44 Class A and Class B GPCRs from 15

distinct neuropeptide GPCR subfamilies57,58. As such, further investigation of these and

other potential GPCR dimer pairs in the Drosophila genome is necessary to determine the

prevalence of dimerization amongst these receptors. Still, given the findings of this study,

along with the wide array of genetic tools available for cell and tissue-specific

manipulation of receptor expression, Drosophila represent an attractive model organism

to investigate the functional roles that GPCR dimerization impacts in multiple

physiologies, and affords the potential to ascribe specific in vivo functional roles to

GPCR oligomeric states and further our understanding of the evolution of GPCR

dimerization amongst the receptor superfamily.

Page 122: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

108

REFERENCES

1. Whitehead, I. P., Zohn, I. E. & Der, C. J. Rho GTPase-dependent transformation

by G protein-coupled receptors. Oncogene 20, 1547–1555 (2001).

2. Hanlon, C. D. & Andrew, D. J. Outside-in signaling – a brief review of GPCR

signaling with a focus on the Drosophila GPCR family. J Cell Sci 128, 3533–3542

(2015).

3. Tautermann, C. S. GPCR structures in drug design, emerging opportunities with

new structures. Bioorg. Med. Chem. Lett. 24, 4073–4079 (2014).

4. White, J. H. et al. Heterodimerization is required for the formation of a functional

GABA(B) receptor. Nature 396, 679–682 (1998).

5. Zhang, X. C., Liu, J. & Jiang, D. Why is dimerization essential for class-C GPCR

function? New insights from mGluR1 crystal structure analysis. Protein Cell 5,

492–5 (2014).

6. Terrillon, S. & Bouvier, M. Roles of G-protein-coupled receptor dimerization

From ontogeny to signalling regulation. EMBO Rep. 5, 30–34 (2004).

7. Franco, R., Martínez-Pinilla, E., Lanciego, J. L. & Navarro, G. Basic

pharmacological and structural evidence for class A G-protein-coupled receptor

heteromerization. Frontiers in Pharmacology vol. 7 (2016).

8. Herrick-Davis, K. Functional significance of serotonin receptor dimerization. Exp.

Brain Res. 230, 375–386 (2013).

9. Tabor, A. et al. Visualization and ligand-induced modulation of dopamine receptor

Page 123: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

109

dimerization at the single molecule level. Sci. Rep. 6, (2016).

10. Portoghese, P. S. From models to molecules: Opioid receptor dimers, bivalent

ligands, and selective opioid receptor probes. J. Med. Chem. 44, 2259–2269

(2001).

11. Pfeiffer, M. et al. Homo- and heterodimerization of somatostatin receptor

subtypes. Inactivation of sst(3) receptor function by heterodimerization with

sst(2A). J. Biol. Chem. 276, 14027–36 (2001).

12. Michineau, S., Alhenc-Gelas, F. & Rajerison, R. M. Human bradykinin B2

receptor sialylation and N-glycosylation participate with bisulfide bonding in

surface receptor dimerization. Biochemistry 45, 2699–2707 (2006).

13. Milligan, G., Ward, R. J. & Marsango, S. GPCR homo-oligomerization. Current

Opinion in Cell Biology vol. 57 40–47 (2019).

14. Meral, D. et al. Molecular details of dimerization kinetics reveal negligible

populations of transient µ-opioid receptor homodimers at physiological

concentrations. Sci. Rep. 8, 7705 (2018).

15. Vischer, H. F., Castro, M. & Pin, J.-P. G Protein-Coupled Receptor Multimers: A

Question Still Open Despite the Use of Novel Approaches. Mol. Pharmacol. Mol

Pharmacol 88, 561–571 (2015).

16. Sakai, T. et al. Evidence for differential regulation of GnRH signaling via

heterodimerization among GnRH receptor paralogs in the protochordate, Ciona

intestinalis. Endocrinology 153, 1841–1849 (2012).

Page 124: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

110

17. Ferré, S. et al. G Protein–Coupled Receptor Oligomerization Revisited: Functional

and Pharmacological Perspectives. Pharmacol. Rev. 66, 413 LP – 434 (2014).

18. Herrick-Davis, K., Weaver, B. A., Grinde, E. & Mazurkiewicz, J. E. Serotonin 5-

HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time

visualization with confocal fluorescence resonance energy transfer. J. Biol. Chem.

281, 27109–16 (2006).

19. Herrick-Davis, K. et al. Native Serotonin 5-HT 2C Receptors Are Expressed as

Homodimers on the Apical Surface of Choroid Plexus Epithelial Cells. Mol.

Pharmacol. Mol Pharmacol 87, 660–673 (2015).

20. Herrick-Davis, K., Grinde, E. & Mazurkiewicz, J. E. Biochemical and biophysical

characterization of serotonin 5-HT2C receptor homodimers on the plasma

membrane of living cells. Biochemistry 43, 13963–13971 (2004).

21. Herrick-Davis, K., Grinde, E., Harrigan, T. J. & Mazurkiewicz, J. E. Inhibition of

serotonin 5-hydroxytryptamine2c receptor function through heterodimerization:

receptor dimers bind two molecules of ligand and one G-protein. J. Biol. Chem.

280, 40144–51 (2005).

22. Park, D. et al. Interaction of structure-specific and promiscuous G-protein-coupled

receptors mediates small-molecule signaling in Caenorhabditis elegans. Proc. Natl.

Acad. Sci. U. S. A. 109, 9917–9922 (2012).

23. Mezler, M., Müller, T. & Raming, K. Cloning and functional expression of

GABAB receptors from Drosophila. Eur. J. Neurosci. 13, 477–486 (2001).

Page 125: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

111

24. Busnelli, M., Mauri, M., Parenti, M. & Chini, B. Analysis of GPCR Dimerization

Using Acceptor Photobleaching Resonance Energy Transfer Techniques. Methods

Enzymol. 521, 311–327 (2013).

25. Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. & Johnson, E. C. Unexpected

role of a conserved domain in the first extracellular loop in G protein-coupled

receptor trafficking. Biochem. Biophys. Res. Commun. 503, 1919–1926 (2018).

26. Kubrak, O. I., Lushchak, O. V., Zandawala, M. & Nässel, D. R. Systemic

corazonin signalling modulates stress responses and metabolism in Drosophila.

Open Biol. 6, 160152 (2016).

27. Sha, K. et al. Regulation of ethanol-related behavior and ethanol metabolism by

the corazonin neurons and corazonin receptor in Drosophila melanogaster. PLoS

One 9, (2014).

28. Johnson, E. C. et al. A novel diuretic hormone receptor in Drosophila: evidence

forconservation of CGRP signaling. J. Exp. Biol. 208, 1239–1246 (2005).

29. Cornea, A. & Michael Conn, P. Measurement of changes in fluorescence

resonance energy transfer between gonadotropin-releasing hormone receptors in

response to agonists. Methods 27, 333–339 (2002).

30. Cheung, T. C. & Hearn, J. P. Dimerizations of the wallaby gonadotropin-releasing

hormone receptor and its splice variants. Gen. Comp. Endocrinol. 144, 280–288

(2005).

31. Pfeiffer, M. et al. Heterodimerization of substance P and mu-opioid receptors

Page 126: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

112

regulates receptor trafficking and resensitization. J. Biol. Chem. 278, 51630–7

(2003).

32. Garcia-Recio, S. & Gascón, P. Biological and Pharmacological Aspects of the

NK1-Receptor. BioMed Research International vol. 2015 (2015).

33. Dinger, M. C., Bader, J. E., Kobor, A. D., Kretzschmar, A. K. & Beck-Sickinger,

A. G. Homodimerization of neuropeptide y receptors investigated by fluorescence

resonance energy transfer in living cells. J. Biol. Chem. 278, 10562–71 (2003).

34. Luquet, S., Perez, F. A., Hnasko, T. S. & Palmiter, R. D. NPY/AgRP neurons are

essentials for feeding in adult mice but can be ablated in neonates. Science (80-. ).

310, 683–685 (2005).

35. Song, G. J., Jones, B. W. & Hinkle, P. M. Dimerization of the thyrotropin-

releasing hormone receptor potentiates hormone-dependent receptor

phosphorylation. Proc. Natl. Acad. Sci. U. S. A. 104, 18303–8 (2007).

36. Hanyaloglu, A. C., Seeber, R. M., Kohout, T. A., Lefkowitz, R. J. & Eidne, K. A.

Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH)

receptor subtypes: Differential regulation of β-arrestins 1 and 2. J. Biol. Chem.

277, 50422–50430 (2002).

37. Lin, T.-Y., Huang, W.-L., Lee, W.-Y. & Luo, C.-W. Identifying a Neuromedin U

Receptor 2 Splice Variant and Determining Its Roles in the Regulation of

Signaling and Tumorigenesis In Vitro. PLoS One 10, e0136836 (2015).

38. Brighton, P. J., Szekeres, P. G. & Willars, G. B. Neuromedin U and its receptors:

Page 127: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

113

Structure, function, and physiological roles. Pharmacological Reviews vol. 56

231–248 (2004).

39. Yurgel, M. E. et al. A single pair of leucokinin neurons are modulated by feeding

state and regulate sleep–metabolism interactions. PLoS Biol. 17, (2019).

40. Al-Anzi, B. et al. The leucokinin pathway and its neurons regulate meal size in

Drosophila. Curr. Biol. 20, 969–978 (2010).

41. Jiang, H. et al. Natalisin, a tachykinin-like signaling system, regulates sexual

activity and fecundity in insects. (2013) doi:10.1073/pnas.1310676110.

42. Bachtel, N. D., Hovsepian, G. A., Nixon, D. F. & Eleftherianos, I. Allatostatin C

modulates nociception and immunity in Drosophila. Sci. Rep. 8, 7501 (2018).

43. Díaz, M. M., Schlichting, M., Abruzzi, K. C., Long, X. & Rosbash, M.

Allatostatin-C/AstC-R2 Is a Novel Pathway to Modulate the Circadian Activity

Pattern in Drosophila. Curr. Biol. 29, 13-22.e3 (2019).

44. Price, M. D. et al. Drosophila melanogaster flatline encodes a myotropin

orthologue to Manduca sexta allatostatin. in Peptides vol. 23 787–794 (2002).

45. Brown, M. R. et al. Identification of a Drosophila brain-gut peptide related to the

neuropeptide Y family. Peptides 20, 1035–1042 (1999).

46. Lee, G., Bahn, J. H. & Park, J. H. Sex- and clock-controlled expression of the

neuropeptide F gene in Drosophila. Proc. Natl. Acad. Sci. 103, 12580–12585

(2006).

47. Chung, B. Y. et al. Drosophila Neuropeptide F Signaling Independently Regulates

Page 128: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

114

Feeding and Sleep-Wake Behavior. Cell Rep. 19, 2441–2450 (2017).

48. Wen, T., Parrish, C. A., Xu, D., Wu, Q. & Shen, P. Drosophila neuropeptide F and

its receptor, NPFR1, define a signalling pathway that acutely modulates alcohol

sensitivity. Proc. Natl. Acad. Sci. U. S. A. 102, 2141–2146 (2005).

49. Zornik, E., Paisley, K. & Nichols, R. Neural transmitters and a peptide modulate

Drosophila heart rate. Peptides 20, 45–51 (1999).

50. Ormerod, K. G. et al. Characterizing the physiological and behavioral roles of

proctolin in Drosophila melanogaster. J. Neurophysiol. 115, 568 (2016).

51. Stern, P. S. et al. Molecular modeling of the binding of pheromone biosynthesis

activating neuropeptide to its receptor. J. Insect Physiol. 53, 803–818 (2007).

52. Alfa, R. W. et al. Suppression of Insulin Production and Secretion by a Decretin

Hormone. Cell Metab. 21, 323–334 (2015).

53. Walsh, S. M. et al. Single Proteoliposome High-Content Analysis Reveals

Differences in the Homo-Oligomerization of GPCRs. Biophys. J. 115, 300–312

(2018).

54. Pioszak, A. A., Harikumar, K. G., Parker, N. R., Miller, L. J. & Xu, H. E. Dimeric

arrangement of the parathyroid hormone receptor and a structural mechanism for

ligand-induced dissociation. J. Biol. Chem. 285, 12435–44 (2010).

55. Hiller, C., Kühhorn, J. & Gmeiner, P. Class A G-Protein-Coupled Receptor

(GPCR) Dimers and Bivalent Ligands. J. Med. Chem. 56, 6542–6559 (2013).

56. Furness, S. G. B. et al. Ligand-Dependent Modulation of G Protein Conformation

Page 129: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

115

Alters Drug Efficacy. Cell 167, 739-749.e11 (2016).

57. Hewes, R. S. & Taghert, P. H. Neuropeptides and neuropeptide receptors in the

Drosophila melanogaster genome. Genome Res. 11, 1126–42 (2001).

58. Johnson, E. C. et al. Identification of Drosophila neuropeptide receptors by G

protein-coupled receptors-beta-arrestin2 interactions. J. Biol. Chem. 278, 52172–8

(2003).

59. Cazzamali, G., Saxild, N. P. E. & Grimmelikhuijzen, C. J. P. Molecular cloning

and functional expression of a Drosophila corazonin receptor. Biochem. Biophys.

Res. Commun. 298, 31–36 (2002).

60. Radford, J. C., Davies, S. A. & Dow, J. A. T. Systematic G-protein-coupled

receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with

novel roles. J. Biol. Chem. 277, 38810–7 (2002).

61. Park, Y., Kim, Y.-J. & Adams, M. E. Identification of G protein-coupled receptors

for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory

of ligand-receptor coevolution. Proc. Natl. Acad. Sci. 99, 11423–11428 (2002).

62. Garczynski, S. F., Brown, M. R., Shen, P., Murray, T. F. & Crim, J. W.

Characterization of a functional neuropeptide F receptor from Drosophila

melanogaster. Peptides 23, 773–780 (2002).

63. Kreienkamp, H.-J. et al. Functional annotation of two orphan G-protein-coupled

receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by

reverse pharmacology. J. Biol. Chem. 277, 39937–43 (2002).

Page 130: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

116

Figure 1: Demonstration of acceptor-photobleaching FRET assay. GPCRs with C-

terminal CFP and YFP fluorophore tags are co-expressed at the plasma membrane of

HEK-293T cells. If the receptors do not form a dimeric complex, the CFP and YFP

fluorophores are separated by greater than 100 angstroms, and no FRET response occurs

(Fig. 1A). If the two receptors assemble as a dimer, CFP and YFP should be located

within 100 angstroms of one another, and thus a FRET response occurs, with some

energy from the excited CFP fluorophore being transferred to the acceptor YFP molecule,

resulting in YFP emission at ~527nm (Fig. 1B). Positive results can be confirmed

through photobleaching the acceptor YFP molecule (Fig. 1C), which ablates the acceptor

YFP fluorophore and “dequenches” the CFP molecule, resulting in an increase in CFP

emission following YFP photobleaching.

Page 131: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

117

Figure 2: Verification of experimental system. Empty pcDNA3 CFP and YFP vectors,

along with C-terminally CFP and YFP tagged 5HT2c receptors, were utilized as negative

and positive controls, respectively. Negligible FRET was observed following acceptor

photobleaching when empty CFP and YFP vectors were coexpressed (4.92%±1.28) (Fig.

Page 132: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

118

2A-F), while the 5HT2c-CFP and 5HT2c-YFP receptor pair exhibited robust FRET

(15.65%±1.72) (Fig. 2G-L) consistent with previous studies. These results suggest the

experimental setup utilized herein accurately differentiates both positive and negative

FRET responses.

Page 133: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

119

Page 134: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

120

Figure 3: Multiple Drosophila Class A neuropeptide receptors exhibit FRET

responses consistent with homodimerization. Seven Drosophila neuropeptide

receptors, AstC-R2 (Fig. 3A-B), CRZR (Fig. 3C-D), LKR (Fig. 3E-F), TKR86C (Fig.

3G-H), NPFR (Fig. 3I-J), PK1R (Fig. 3M-N), and ProcR (Fig. 3O-P) were C-terminally

tagged with CFP or YFP fluorophores and co-expressed in HEK293T cells to evaluate

potential homodimer pairs. Significant increases in CFP intensity following acceptor

photobleaching, indicative of FRET, were observed for each homodimer pair tested (Fig.

3Q) Co-expression of NPFR-CFP and TKR86C-YFP as donor and acceptor, respectively,

Page 135: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

121

exhibited negligible FRET (Fig. 3K-L), (5.80%±1.81, n=10) that was not significantly

different from the CFP-YFP negative control (Fig. 2A-F; P<.05), indicating negligible

protein-protein interactions between these two distantly related receptors. These results

suggest that the receptors assayed in this study assemble as homodimers when expressed

in living cells. Black bars represent data that were significantly different from the CFP-

YFP negative control (P<.05).

Page 136: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

122

Receptor Family Accession# Reference

ProcR TRH CG6986 Johnson et al., 200358

CrzR GnRH CG10698

Cazzamali et al., 2002; Johnson et al.,

200358,59

LKR Tachykinin CG10626 Radford et al., 200260

PK1R NMU CG9918 Park et al., 200261

NPFR NPY CG1147 Garczynski et al., 200262

AstC-R2 Somatostatin CG13702

Kreienkamp et al., 2002; Johnson et

al., 200358,63

TKR86C Tachykinin CG6515 Johnson et al., 200358

Table 1: Receptors utilized in FRET dimer screen. Each of the above receptors had

been previously characterized and cloned into pcDNA3 or pcDNA5 expression vectors.

Family assignments were based on receptor homology to vertebrate GPCRs. Vertebrate

homologs of each Drosophila receptor listed have previously been reported to form either

homo- or heterodimers when expressed in a heterologous system.

Page 137: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

123

Receptor Forward Primer Reverse Primer

Forward

Restriction Site

Reverse

Restriction

Site

LKR GCAAGCTTATGGCAATGGACTTAATCGAGCAG CTCGAGAAGTGGTTGCCACAAGGACTTGCC HindIII XhoI

CRZR GCAAGCTTATGGAGGACGAGTGGGGCTCCTTT CTCGAGCTGCACTGGAAGCACTTGGAGCTC HindIII XhoI

ProcR GCAAGCTTATGACAATGTCCTCGACGTCGACA CTCGAGCGCTATCAGGCGACCCGTATTACG HindIII XhoI

TKR86C GCAAGCTTATGTCGGAGATTGTCGACACCGAG GCGGCCGCAACATCTGCTTGGGACTGAGCT HindIII NotI

PK1R GCAAGCTTATGTCCGCTGGCAATATGAGCCAT CTCGAGGTTGACTTGGACACCGATCATGGC HindIII XhoI

NPFR GCAAGCTTATGATAATCAGCATGAATCAGACG CTCGAGCCGCGGCATCAGCTTGGTGACCTC HindIII XhoI

AstC-R2 GCAAGCTTATGGAAGGTGGATGGTGGCGAGGA CTCGAGTAAGTCCGTGTGGAGCACGGGCGG HindIII XhoI

Table 2: List of primers used for directional cloning of receptor cDNA into pcDNA3

CFP or pcDNA3 YFP expression vectors. For each receptor, stop codons were removed

from reverse primers to allow for expression of YFP and CFP C-terminally tagged

receptors. Sequences for the restriction sites HindIII and XhoI were added via PCR to

facilitate directional cloning into final expression vectors for each receptor used except

for TKR86C, where sequences for HindIII and NotI were added due to an internal XhoI

recognition sequence present in the cDNA for this receptor.

Page 138: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

124

Figure S1: Verification of signaling in fluorophore tagged receptors. YFP tagged

receptors for which peptides were readily available (LKR and CRZR) were assayed to

determine whether the addition of a C-terminal fluorophore tag impacted receptor

signaling. YFP tagged receptors were compared against wild type variants of each

receptor. Each receptor variant was challenged with either 10-6 M ligand or vehicle (n=3)

and signaling was measured through luciferase activity generated by the SRE-luc

reporter. No significant differences were seen across all receptors tested, with LKR-YFP

showing 3.13-fold induction over vehicle and CRZR-YFP showing 2.90-fold induction

over vehicle, while their respective wild type receptors exhibited 3.41-fold induction over

vehicle (LKR WT) and 3.11-fold induction over vehicle (CRZR WT). These data show

that the addition of a C-terminal fluorophore tag to these receptors does not compromise

receptor function.

0

0.5

1

1.5

2

2.5

3

3.5

4

LKR WT LKR YFP CRZR WT CRZR YFP

Fo

ld i

nd

uct

ion

ov

er

ve

hic

le

Receptor

Page 139: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

125

CHAPTER IV: Conclusions and future directions

The goal of this dissertation was to elucidate specific mechanisms underlying

GPCR signaling and function. Using a combination of approaches, I have uncovered a

novel role for a highly conserved amino acid motif in GPCR function, as well as

expanded our knowledge of GPCR dimerization by providing the first evidence of such

an event occurring in Drosophila. The combination of these findings also extends our

knowledge of GPCR evolution, and suggests that many aspects of receptor function arose

early in the evolution of the receptor family.

The second chapter of this dissertation specifically explored the prevalence and

functional role of an amino acid motif present on the vast majority of Class A GPCRs

first extracellular loop, the WxFG motif. While this motif had been previously identified,

a full exploration of the conservation of this motif across Class A GPCR subfamilies had

not been performed prior to our research. Our bioinformatics analysis showed that this

motif is conserved in 17 of the 19 Class A GPCR subfamilies, in addition to being

present in ~90% of all Class A GPCRs1. By generating mutant receptor variants for

multiple Class A subfamily members, sourced from a variety of vertebrate and

invertebrate taxa, we showed that the presence of an aromatic amino acid at the W

position in the WxFG motif is necessary for wild type receptor signaling. Additionally,

we suggest an alternative mechanism for the loss of receptor function following

mutagenesis of this residue than had been previously put forward. Previous work on this

amino acid motif by Klco et al suggested that mutagenesis of the conserved tryptophan

residue generated receptor mutants which were able to bind ligand, but unable to

appropriately translate ligand binding into specific cellular responses2. Our findings

Page 140: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

126

suggest that, instead, such receptor manipulations lead to defective GPCR trafficking,

resulting in these receptors not reaching the cell surface and instead being retained in the

ER-Golgi complex. The findings of previous investigations can be reconciled with our

novel mechanism through a comparison of the methods used in the respective studies –

Klco et al performed ligand binding assays on membrane preparations of cells expressing

both wild type and mutant receptors. Such preparations would have captured not only

receptors present at the plasma membrane, but also receptors retained in the ER-Golgi

complex. As such, even though their group found that mutant receptors were able to bind

ligand, we suggest that, in the actual environment of the cell, these receptors never reach

the cell surface, and are thus unable to interact with their specific ligands, resulting in the

lack of signaling responses noted in all studies of this amino acid motif. As such, our

findings offer a novel mechanism by which this motif contributes to GPCR function.

The third chapter of this dissertation investigated whether Drosophila GPCRs,

specifically those involved in neuropeptide recognition and signaling, assembled as

dimers at the plasma membrane. While GPCR dimerization has been widely explored

over the past two decades, with a multitude of GPCR homo- and heterodimeric entities

identified and described, the vast majority of these studies looked specifically at

vertebrate GPCRs, while invertebrate GPCR dimerization remains a neglected field of

study3,4. Through C-terminal tagging of Drosophila neuropeptide GPCRs with CFP and

YFP fluorophores, we were able to utilize an acceptor-photobleaching FRET

methodology to show that multiple Drosophila receptors assemble as homodimers at the

plasma membrane. These findings are significant, as they are the first to identify any

GPCR dimerization in Drosophila. Additionally, by focusing on receptors previously

Page 141: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

127

shown to assemble as higher order structures in other taxa, our results suggest that

receptor dimerization has been conserved throughout the evolution of specific receptor

subtypes, rather than being a taxa-specific phenomenon. It is therefore likely that

dimerization arose early in the evolution of the receptor superfamily, and is likely

critically important for wild-type receptor function in many GPCRs.

Although this work has shed light on mechanisms underlying GPCR function and

assembly, there is much left that could be explored. I have clearly shown that the WxFG

motif plays a critical role in the appropriate cell surface trafficking of a multitude of

Class A GPCRs. However, as this was explored using a heterologous expression system,

the question as to whether similar phenotypes occur in vivo, where other cellular

machinery such as chaperone proteins may assist in GPCR trafficking, remains

unresolved. As such, a logical next step would be to perform these same mutagenic

manipulations via CRISPR or similar methodology in the genome to determine whether a

similar trafficking defect occurs. Such an effort would be best focused on the subset of

Drosophila receptors studied, given the genetic tools available in this model organism.

These manipulations could also be used as loss of function alleles to further study the

roles of these receptors in a multitude of behaviors and physiologies.

Additionally, my work on dimerization of Drosophila GPCRs suggests that many

neuropeptide receptors in the fly are capable of assembling as homodimers, however, this

was verified solely using FRET microscopy. To increase confidence in my findings, a

logical next step would be to utilize another method to detect GPCR dimers, such as Co-

IP or BiFC, and see if similar results were obtained. Additionally, generating UAS

constructs of CFP and YFP tagged receptors used in this study, or simply generating CFP

Page 142: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

128

and YFP fusion proteins in the genome through CRISPR, would allow one to assess

whether dimerization of these receptors occurs in vivo. Such a finding would add to the

dearth of in vivo dimerization studies in the extant literature. Also, my work focused

solely on establishing homodimerization of Drosophila GPCRs, it would be interesting to

further screen receptors to determine whether disparate GPCRs were capable of

assembling as heterodimers, and if so, what impact heterodimerization has on receptor

function. These experiments would provide valuable information regarding both the

extent and conservation of dimerization across taxa, as well as further elucidating the

functional roles of dimerization in receptor signaling in an in vivo setting.

In conclusion, my work has furthered our fundamental understanding of multiple

aspects of GPCR function. I have presented evidence supporting a novel mechanism for a

highly conserved amino acid motif in receptor trafficking to the cell membrane.

Additionally, I have shown that multiple Drosophila GPCRs from disparate receptor

subfamilies are capable of dimeric assembly at the plasma membrane, adding to our

limited knowledge of invertebrate GPCR dimerization, while also furthering our

understanding of the conservation of dimerization throughout the evolution of the

receptor superfamily. Together, these investigations provide valuable insight into

multiple aspects of GPCR function, while also offering additional evidence to support the

obsolescence of the classical two-state model.

Page 143: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

129

REFERENCES

1. Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. & Johnson, E. C. Unexpected

role of a conserved domain in the first extracellular loop in G protein-coupled

receptor trafficking. Biochem. Biophys. Res. Commun. 503, 1919–1926 (2018).

2. Klco, J. M., Nikiforovich, G. V & Baranski, T. J. Genetic Analysis of the First and

Third Extracellular Loops of the C5a Receptor Reveals an Essential WXFG Motif

in the First Loop. J. Biol. Chem. 281, 12010–12019 (2006).

3. Sakai, T. et al. Evidence for differential regulation of GnRH signaling via

heterodimerization among GnRH receptor paralogs in the protochordate, Ciona

intestinalis. Endocrinology 153, 1841–1849 (2012).

4. Park, D. et al. Interaction of structure-specific and promiscuous G-protein-coupled

receptors mediates small-molecule signaling in Caenorhabditis elegans. Proc. Natl.

Acad. Sci. U. S. A. 109, 9917–9922 (2012).

Page 144: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

130

Michael J. Rizzo [email protected]

EDUCATION

Wake Forest University, Winston-Salem, NC August 2011 – Present Ph.D. Candidate in Biology; GPA: 3.75

University of Pittsburgh School of Medicine, Pittsburgh, PA July 2009 – June 2010 Ph.D. Student in Biomedical Sciences; GPA: 3.64 University of Virginia, Charlottesville, VA August 2004 – May 2008 B.A. in Biology, Minor in Philosophy; GPA: 3.34

• Phi Eta Sigma National Honor Society (2005) • Dean’s List (2005)

PROFESSIONAL EXPERIENCE

High Point University, Department of Biology, High Point, NC August 2018 – Present Instructor of Biology

• Taught multiple lecture and laboratory courses. • Helped develop new syllabus and course objectives for non-majors course • Served in multiple department community outreach events

Wake Forest University, Department of Biology, Winston-Salem, NC August 2011 – Present Graduate Research and Teaching Assistant

• Taught laboratory courses in a variety of biological subdisciplines. • Undertook research on G-protein coupled receptors (GPCRs) exploring various aspects of their biology, examining receptors from both Drosophila and humans. • Served as Graduate representative to University Honor Council. • Presented posters and talks at multiple institutional meetings, as well as Genetics Society of America and Cold Spring Harbor Laboratories international conferences. • Mentored a variety of undergraduate students in molecular biology and genetic techniques. • Wrote and edited multiple grant applications, secured independent funding for research. • Served as reviewer for multiple publications in scientific journals.

Galax City Public Schools, Galax, VA November 2010 – April 2011 Substitute Teacher

• Performed teaching duties as needed for a variety of ages. Classes included science, reading, and special education.

Darden/Curry Partnership for Leaders in Education, Charlottesville, VA October 2010 – April 2011 Lead Editor

• Edited collection of case studies for publication, “District Case Studies and Individual Lessons in Leadership,” Dan Duke, Eleanor Smalley.

Cardiovascular Research Center, University of Virginia, Charlottesville, VA October 2007 – June 2009 Research Assistant

• Performed an array of laboratory related techniques, including western blots and survival and non- survival surgery on mice.

Page 145: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

131

• Actively involved in research pertaining to connexin isoforms found in the vasculature, including editing potential publications and creating presentations on the subject matter. • Performed cell culture work and sterile procedure. • Designed and maintained laboratory website

General Clinical Research Center, UVA Hospital, Charlottesville, VA April 2006 – August 2006 Computer Technician

• Performed multiple and complex IT support, including detecting bugs and general compatibility issues. • Implemented state of the art software and hardware applications. • Accountable for general computer maintenance.

SKILLS

Photoshop, Graphpad Prism, Microsoft Office, ImageJ, Carl Zeiss Zen, Tissue Culture, Drosophila and mouse husbandry/dissections, Molecular Cloning, Bioinformatic Sequence Analysis, PCR, RNA and DNA Isolation, Plasmid Prep, Western Blot, DNA Transfection, Phenol-Chloroform Extraction, rudimentary Java

GRANTS AND AWARDS

Center for Molecular and Cell Signaling graduate fellowship – $25,000 2013 – 2014

TEACHING EXPERIENCE

Principles of Cell Biology – BIO1500/BIO1501 2018 – 2019 Biology: A Human Perspective – BIO1100 2018 – 2019 Molecular Biology and Genetics Lab – BIOL213 2014 – 2018 Cell Biology Lab – BIOL214 2012 – 2013 Comparative Physiology Lab – BIOL114 2011 – 2012

UNDERGRADUATE STUDENTS MENTORED

Kandis McNeil 2017 – 2018 Jack Evans; Undergraduate Honors Award 2017 – 2018 Karleigh Smith 2017 – 2018 Harriet Hall 2016 – 2018 Kevin Robinson 2016 Morgan Burt; Undergraduate Honors Award, Carolina Biological Outstanding 2012 – 2015

Undergraduate Research Award Donika Hasanaj 2014 Cole Crowson; Undergraduate Honors Award 2012 – 2014 Rebecca Perry; Undergraduate Honors Award, Carolina Biological Outstanding 2011 – 2013

Undergraduate Research Award, Cocke Outstanding Student Scholar Award. Brian Vega 2012

Page 146: MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR ...€¦ · FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan ... microscopy to determine both the

132

PUBLICATIONS

Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. & Johnson, E. C. (2018) Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking. Biochem. Biophys. Res. Commun. 503, 1919–1926

Miller, M.R., Mandell, J.B., Beatty, K.M., Harvey, S.A.K., Rizzo, M.J., Previte, D.M., Thorne, S.H., and McKenna, K.C. (2014). Splenectomy promotes indirect elimination of intraocular tumors by CD8+ T cells that is associated with IFNγ- and Fas/FasL-dependent activation of intratumoral macrophages. Cancer Immunol Res 2, 1175–1185.

Straub, A.C., Johnstone, S.R., Heberlein, K.R., Rizzo, M.J., Best, A.K., Boitano, S., and Isakson, B.E. (2010). Site-specific connexin phosphorylation is associated with reduced heterocellular communication between smooth muscle and endothelium. J. Vasc. Res. 47, 277–286.

Johnstone, S.R., Ross, J., Rizzo, M.J., Straub, A.C., Lampe, P.D., Leitinger, N., and Isakson, B.E. (2009). Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation. Am. J. Pathol. 175, 916–924.

POSTERS AND PRESENTATIONS

Poster: Molecular dissection of Drosophila G protein-coupled receptor oligomerization Michael Rizzo, Erik Johnson Neurobiology of Drosophila, Cold Spring Harbor Laboratories (2015) Presentation: Molecular dissection of Drosophila G protein-coupled receptor oligomerization Michael Rizzo Wake Forest University Center for Molecular Communication and Signaling (2015) Poster: Elucidation of Drosophila melanogaster G protein-coupled receptor interactions through heterodimerization and chimeric receptor studies. Michael Rizzo, Erik Johnson Genetics Society of America (2013)