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ARTICLE IN PRESSG ModelSCDB-1903; No. of Pages 10

Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

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eedback regulation of G protein-coupled receptor signalingy GRKs and arrestins

oseph B. Blacka, Richard T. Premontb, Yehia Daakaa,∗

Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610, United StatesDepartment of Medicine, Duke University Medical Center, Durham, NC 27710, United States

r t i c l e i n f o

rticle history:eceived 23 October 2015ccepted 19 December 2015vailable online xxx

a b s t r a c t

GPCRs are ubiquitous in mammalian cells and present intricate mechanisms for cellular signalingand communication. Mechanistically, GPCR signaling was identified to occur vectorially through het-erotrimeric G proteins that are negatively regulated by GRK and arrestin effectors. Emerging evidencehighlights additional roles for GRK and Arrestin partners, and establishes the existence of interconnectedfeedback pathways that collectively define GPCR signaling. GPCRs influence cellular dynamics and can

eywords:PCR

proteinrrestin

mediate pathologic development, such as cancer and cardiovascular remolding. Hence, a better under-standing of their overall signal regulation is of great translational interest and research continues toexploit the pharmacologic potential for modulating their activity.

© 2016 Elsevier Ltd. All rights reserved.
RKignal transductioniased signaling
ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. 7TMR/GPCR signaling overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.1. Diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Structure and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. G protein signaling mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. G protein classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5. GPCR functional classification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00

3. 7TMR/GPCR dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.1. Receptor desensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Receptor sequestration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Receptor downregulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.4. Constitutive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.5. Agonist and antagonist dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Signal termination: GRK, arrestin, and G protein switching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1. Regulated GPCR-G protein coupling and G protein switching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2. GRK signal termination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .004.3. Arrestin signal termination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.4. Arrestin GPCR feedback regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. GRK and arrestin-mediated signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.1. Non-canonical GRK activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.2. Arrestin-mediated signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: J.B. Black, et al., Feedback regulatioSemin Cell Dev Biol (2016), http://dx.doi.org/10.1016/j.semcdb.2015.1

5.3. GPCR biased signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author.E-mail address: [email protected] (Y. Daaka).

ttp://dx.doi.org/10.1016/j.semcdb.2015.12.015084-9521/© 2016 Elsevier Ltd. All rights reserved.

n of G protein-coupled receptor signaling by GRKs and arrestins,2.015

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dx.doi.org/10.1016/j.semcdb.2015.12.015


http://www.sciencedirect.com/science/journal/10849521

http://www.elsevier.com/locate/semcdb

mailto:[email protected]


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ARTICLESCDB-1903; No. of Pages 10

J.B. Black et al. / Seminars in Cell & D

. Introduction

Cellular differentiation is a hallmark of multicellular orga-isms that is essential for specialization at the cell, tissue, andrgan level. Specialization promotes organismal development andurvival by permitting cells to differentiate into cellular lin-ages that possess unique properties, such as sensation, secretion,bsorption, or conductivity [1]. Multicellular organisms coordinatehese activities to sustain homeostasis. Cell surface receptors andignaling molecules represent a pivotal mechanism for overcom-ng this challenge by facilitating cellular communication [2,3]. Theeven Transmembrane Span Receptors (7TMR)/G Protein-Coupledeceptors (GPCRs) family represents the largest superfamily ofell surface receptors, with over 800 distinct receptors encodedithin the human genome [4]. This review highlights the biologic

ole and regulation of 7TMR/GPCR and their interacting proteins,hile reviewing their structure, classification, and mechanism

f action.

. 7TMR/GPCR signaling overview

.1. Diversity

7TMR/GPCRs are activated by diverse extracellular signals, suchs proteins, peptides, lipids, small molecules, ions, and photons.hese receptors transduce extracellular cues into intracellular sig-als to facilitate cell responses. 7TMR/GPCRs and their diversitynderpin their ability to regulate highly varied physiological func-ions responsible for sensation, adaptation, and survival [4–7]. Inhe context of sensation, visual processing is initiated by fourisual 7TMR/GPCRs, gustation uses several sweet, bitter and umamiglutamate) GPCRs; and olfactory sensation is initiated by sev-ral hundred 7TMR/GPCRs [7–10]. In addition, over 150 distinctPCRs and their cognate ligands regulate critical biophysical pro-esses, such as endocrine physiology, immunomodulation, andeurotransmission/neuromodulation. Despite extensive researchver the past 3 decades, over 100 “orphan” receptors remain thatave either no known endogenous ligand or unclear physiological

unctions [11].

.2. Structure and classification

The 7TMR/GPCR family name reflects its members’ structurend function. This receptor family is structurally defined by aeven alpha-helix transmembrane span topology [4,7,12]. Themino terminus and three transmembrane connecting loops areositioned extracellularly, while the carboxy terminus and threedditional transmembrane connecting loops are located intracel-ularly. In addition to structural homology, this receptor familyargely shares a common mechanism of action: GPCR activationnitiates signal transduction across the cell membrane, whichctivates an intracellular heterotrimeric GTP-binding proteinsG proteins) [5].

The 7TMR structural superfamily is divided into three canoni-al receptor families and other non-canonical families [13]. Theseamilies are defined by conserved sequence elements locatedrimarily in the receptor transmembrane region. Overall, theselassifications are subdivided into canonical (family A–C) and non-anonical groupings. Unlike family A–C receptors, non-canonicalTMR appear to primarily utilize mechanisms other than G pro-eins for signal propagation. However, numerous recent reports


rgue that specific non-canonical 7TMR can indeed activate G pro-eins and be regulated like GPCRs, in addition to their non-canonicalctivities [14]. Family A represents the largest canonical subfamilynd is defined by homology with the visual receptor rhodopsin

PRESSmental Biology xxx (2016) xxx–xxx

and the �2-adrenergic receptor (�2AR). Family B is defined bysequence homology to secretin and glucagon receptors. Expres-sion of family B receptors is localized largely to gastrointestinaland neuronal tissue. The smallest canonical 7TMR subfamily, fam-ily C receptors are neuromodulatory metabotropic glutamate andGABAB receptors. This family is expressed in both the peripheraland central nervous systems and is responsible for processingdiverse signals, such as pain, anxiety, and memory formation.Non-canonical receptors include those of the Frizzled family thatrecognize Wnt ligands, or the Hedgehog co-receptor Smoothened.These receptors exert their effect through G protein independentmechanisms [13].

2.3. G protein signaling mechanism

G protein activation is dependent primarily upon GPCR stim-ulation. GPCRs remain mostly inactive on the cell surface untilbound by a cognate activating agonist and initiate G protein activa-tion (Fig. 1). A GPCR’s corresponding G protein generally remainsinactive until GPCR activation. In their inactive conformation, Gproteins exist in a heterotrimeric state: the constituent �-subunit,bound to GDP, is tightly associated with the ��-subunit dimer.GPCRs act as guanine nucleotide exchange factors (GEF) for Gproteins when agonist-activated [15–17]. Consequently, GPCR acti-vation stimulates the exchange of GDP for GTP on the �-subunit.GDP/GTP exchange on the �-subunit stimulates the dissociationof activated �·GTP from the ��-subunits. Activated �·GTP thenbinds to downstream signaling effectors, such as adenylyl cyclaseor phospholipase C, to alter their activity to increase levels ofintracellular second messengers, such as cyclic AMP (cAMP) orinositol tris-phosphate (IP3) [17,18]. In addition to �·GTP, unbound��-subunits bind to and activate their own effectors, including Gprotein-regulated Inward Rectifying K (GIRK) channels (Fig. 1) andprotein kinases, such as GRK2 and PAK [19–24].

For deactivation, the �-subunit encodes an intrinsic GTPase thathydrolyzes GTP to GDP to inactivate its ability to propagate thesignal cascade [15,16]. Additional GTPase-activating proteins accel-erate this deactivation, such as regulator of G protein signaling(RGS) family accessory proteins [25,26]. When GTP bound to an�-subunit is hydrolyzed to GDP, the �·GDP dissociates from itseffectors and re-associates with ��-subunits, thereby reassem-bling an inactive G protein heterotrimer. The dissociation of the�-subunit and ��-subunit from their effectors effectively ends thepropagation of the signal cascade by deactivation of the effector[15–17].

G protein mediated GPCR signaling is a highly amplified sys-tem. Upon activation, GPCR’s guanine nucleotides exchange activitylast long enough to induce activation of numerous individual Gproteins. Activated G proteins provide further signal amplificationowing to �-subunit’s ability to produce many second messengermolecules (e.g., cAMP, IP3) during the time it remains bound toan effector enzyme. Consequently, small amounts of a hormonereleased from a distant part of a multicellular organism can pro-foundly influence the function of a distant target cell.

2.4. G protein classification

G proteins are classified based upon their � subunit structureand function, and fall into four subclasses. Each G protein subclasshas distinct downstream effectors and produces unique signalingeffects on the cell. First, the stimulatory G proteins (Gs) stimulateadenylyl cyclase to convert ATP into cyclic AMP (cAMP). Elevated


cAMP results in the activation of cAMP-regulated proteins, suchas, specific kinases (protein kinase A, PKA), cyclic nucleotide-gatedchannels, and the Epac GEF for Rap small GTP-binding proteins[5,15,16,27]. Second, the inhibitory heterotrimeric Gi proteins


ARTICLE IN PRESSG ModelYSCDB-1903; No. of Pages 10

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echanistically oppose Gs activity by inhibiting adenylyl cyclase,hich reduces intracellular cAMP levels [15,16,28]. Furthermore,

he ��-subunits of Gi proteins can exert cellular effects by regulat-ng GIRK channels and other effectors. Transducins (Gt) representision-specific Gi family members and are stimulated by light-ctivated rhodopsin to regulate retinal cGMP phosphodiesterase29]. Similarly, gustducin (Ggust) is a Gi family member stimu-ated by tastant receptors in taste buds. Third, Gq proteins activatehospholipase C-� enzymes upon stimulation by cognate GPCRs.hospholipase C-� hydrolyzes plasma membrane phosphatidylnositol lipids, releasing IP3 and diacyl glycerol that act to increasentracellular calcium and protein kinase C activity [28]. Fourth, the12 proteins activate GEFs for the RhoA small GTP-binding protein,ctivating it to regulate the cytoskeleton [28,30,31].

.5. GPCR functional classification

In addition to classifications based upon sequence similaritySection 2.2), GPCRs can be categorized by their cognate G proteinnd the downstream pathways they regulate [32]. Interestingly,hile some GPCRs appear to exclusively couple with one class of

protein (e.g., “Gs-coupled receptor” or “Gq-coupled receptor”),ther GPCRs can activate G proteins from two or more classes.ot all G proteins within a class are equivalent: receptors canave a preference for specific subtypes of G proteins within alass. In particular, evidence using mice deficient in individual Giamily members has made it clear that a receptor may couple tonly one member of the Gi subfamily even when other membersre available. This signaling diversity is further increased by theact that there are five �-subunits and twelve �-subunits that canheoretically assemble (with the �-subunits) several hundred com-inations, and both receptors and ��-subunit-activated effectorsan prefer specific � + �� combinations. Despite this complexity,he general principles of G protein mediated signaling hold true28,33].

GPCRs can also be categorized according to their ligands [32,34].


eceptors that bind the same ligand are generally also similarased upon other classification systems. For instance, receptorshat bind the same ligand are usually very highly related andorm distinct subgroups by homology clustering. For example,

second messengers cyclic AMP (cAMP) and inositol tris-phosphate (IP3). Gi�·GTP

receptors for glycoprotein hormones (e.g., LH, FSH) are struc-turally similar to each other, as are receptors for the biogenicamines (e.g., serotonin, dopamine, adrenaline). While many uniqueligand–receptor pairs exist, some endogenous ligands can bindwith high affinity to multiple receptor subtypes (e.g., 12 GPCRs acti-vated by serotonin, 5 by acetylcholine). Overall, this complexity ofGPCRs and ligands increases the specificity of desired target organeffects.

3. 7TMR/GPCR dynamics

As noted, receptors are generally inactive in the absence oftheir activating agonist. Once a specific 7TMR/GPCR is stimu-lated, it activates downstream intracellular signaling pathwaysin response to agonist binding. However, this model vastly over-simplifies the dynamics of 7TMR/GPCR mediated signaling. Thissection seeks to define the mechanisms that regulate the signalingprocess.

3.1. Receptor desensitization

7TMR/GPCR-regulated systems exhibit graded responses uponactivation. The amount of ligand signal defines the downstreamsignal; more agonist promotes more response. Because GPCR sys-tems are highly amplified, a small amount of ligand occupying afraction of receptors may induce a maximal response. However,each receptor system displays a memory of prior activation thatinfluences its ability to be stimulated in the future. The fraction ofreceptors that must be activated to achieve a full effect varies basedon how active the system has been recently. Cells with receptorsthat have been active recently respond by becoming less sensitiveto further stimulation, while cells that have not been stimulatedfor some time become more sensitive to future stimulation. Thisphenomenon is termed desensitization [22,35–37]. Desensitizationserves to prevent overstimulation of target cells in the face of highagonist concentrations (Fig. 2A), while the reverse process of resen-


sitization allows cells to still recognize and respond to low amountsof agonist [38–40]. Overall, these interlinked processes allow a cellto maintain sensitivity to a ligand in conditions of either high andlow signaling tone.


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The ability to alter GPCR sensitivity is critical for cellularesponses such as chemotaxis, wherein a cell responds to a gradientf chemoattractant agonist. Cells must interpret differences in ago-ist concentration that vary by a few percent difference from oneide of the cell to other to effectively migrate toward the source of

ligand, such as a site of infection. Receptor desensitization mech-nisms allow cells to decipher the cues of a shallow ligand gradienthrough differential signaling sensitivity to define the ‘front’ versushe ‘rear’ of a migrating cell [41]. Immune cells utilize such a mecha-ism in order to migrate toward injured tissues following gradientsf tissue-released GPCR ligand chemokines [42].

Overstimulated receptors require higher levels of agonist toroduce equal downstream effects. This is clinically exemplified

n heart failure, wherein poor contractility of the heart drivesatecholamine release to act on cardiac adrenergic receptors toncrease contractility. As heart receptors adapt to the high levels oftimulation, this positive feedback overdrive eventually becomesneffective and actually counterproductive, as it promotes furtherathological remodeling of the heart [28,43,44].

.2. Receptor sequestration

Receptor levels on the cell surface are dynamic and change inesponse to agonist stimulation. Receptors are removed from theell surface upon activation, and return to the cell surface after ago-ist levels subside (Fig. 2B and C) . Short-term endocytotic removalf receptors from the cell surface is termed sequestration [45]. Dif-erent receptor types utilize distinct pathways for removal fromhe cell surface, such as clathrin-coated pits or caveolae-mediatedndocytosis. Once internalized, receptors traffic in intracellularesicles through a series of intracellular vesicular compartmentshat eventually lead to release of the receptor back onto the cellurface in an activatable state [46,47].

.3. Receptor downregulation

Long-term over-activation of a GPCR can lead to a substan-ial reduction in cell surface receptor levels. This is termedown-regulation and can result in substantial reductions ineceptors present at the cell surface (Fig. 2C). Mechanistically,own-regulation is initially comparable to receptor sequestration.owever, unlike sequestration, down-regulation targets internal-

zed receptors for lysosomal degradation. Thus, recovery of normaleceptor level at the cell surface requires new receptor synthesis48,49].

.4. Constitutive activity

Receptors possess varying degrees of agonist-independentctivity, termed constitutive activity, for activating downstreamignaling pathways [50]. Constitutive activity levels are recep-or specific and depend on receptor expression level. Receptorsith low constitutive activity exhibit the highest degree of ligand-

timulated activity, whereas agonist activation of receptors withigh constitutive activity may only promote a two-fold increase

n signal upon agonist stimulation, due to their high basal levelf activity. Constitutive activity is hypothesized to occur as aonsequence of the molecular dynamics of the receptor protein:


eceptors constantly transition among multiple conformations, and subset of these conformations are capable of inducing G proteinctivation, thus producing constitutive activity. A receptor withigh constitutive activity thus has more low-energy states that

avor G protein activation than a receptor with low constitutivectivity [51–54].


3.5. Agonist and antagonist dynamics

GPCR conformational states determine their ability to activatecoupled G proteins. Agonists stabilize active GPCR conformationsto induce signaling, while antagonists stabilize inactive conforma-tional states to prevent signaling. Agonists are classified based uponthe degree to which they activate GPCRs. A full agonist promotesmaximal GPCR activation (maximal as compared to the nativeligand), while a partial agonist activates GPCR activity to a lesserdegree even at the highest concentrations of that ligand. In contrast,an antagonist inhibits activation by either occupying the bindingsite for the natural ligand (orthosteric site) or another site (allostericsite) that precludes subsequent agonist mediated GPCR activation[55,56]. Furthermore, agonists can alter receptor constitutive activ-ity. Inverse or negative agonists silence the activity of constitutivelyactive receptors by suppressing their basal constitutive activity,through preventing spontaneous transitions to active states. In con-trast, neutral antagonists bind a receptor without altering the abilityto transition among inactive and active states, thus permitting basalactivity to continue [57,58]. Overall, molecules that bind to a recep-tor can affect GPCR function along a continuum, from full activationthrough complete inactivation.

4. Signal termination: GRK, arrestin, and G proteinswitching

Termination of GPCR signaling is mediated by multiple mecha-nisms. Signaling at the receptor can be terminated by degrading theligand, as is the case for most peptide agonists, or by removing theligand from the vicinity of the receptor, as through reuptake of thebiogenic amine dopamine from synapses into presynaptic neuronsfor re-storage in vesicles for later release. The intrinsic GTPase activ-ity of G proteins and its acceleration by GTPase-activating proteinsof the RGS protein family (Section 2.3) limits the lifetime of down-stream signaling once an agonist is no longer present at sufficientconcentration at the receptor to drive new G protein activation. Butin addition to these general modes of regulation, GPCRs themselvesare subject to direct regulation by a two part mechanism consistingof G protein-coupled receptor kinases (GRKs) and arrestins. Thesetwo classes of cellular regulatory proteins cooperatively functionto terminate GPCR signaling, as well as by mechanisms that alterreceptor coupling to G proteins.

4.1. Regulated GPCR-G protein coupling and G protein switching

GPCR affinity for specific G protein subtypes is influenced byseveral factors, including receptor structure and posttranslationalmodification. Individual GPCRs may dynamically couple amongstseveral G protein subtypes. Indeed, receptors may experience aphenomena termed “G protein switching”, wherein a GPCR’s affin-ity for a particular G protein subtype alters in response to cellulardynamics, such as receptor posttranslational modifications. The�2AR demonstrates G protein switching, as it dynamically coupleswith both Gs and Gi proteins [33,59]. �2AR G protein switchingoccurs as a negative feedback regulatory mechanism mediated byPKA phosphorylation of the receptor (Fig. 3A). Upon initial stimu-lation, �2AR activates the Gs protein to increase adenylyl cyclasesignaling and PKA activity to propagate the cellular response. Inaddition to eliciting second messenger responses, PKA phospho-rylates site-specific residues on �2AR [60]. PKA phosphorylation


of �2AR alters the receptor coupling from Gs to Gi, and acti-vated Gi subsequently deactivates Gs protein and results in adecrease in cAMP production and PKA activity [33,61]. Effectively,PKA phosphorylation of �2AR and G protein switching mediates a


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egative feedback loop with respect to the initial Gs signal activa-ion (Fig. 3A).

.2. GRK signal termination

GPCR kinases (GRKs) are protein serine/threonine kinases of therotein kinase A/G/C subfamily. This kinase family initiates GPCRignal termination by phosphorylating ligand-bound GPCRs. Theseinases recognize the active conformation of GPCRs and phospho-ylate serine and threonine residues within the carboxy terminalail and third intracellular loop of the receptor. GPCR phosphoryla-ion itself generally does not decouple the activated GPCR from itsognate G protein. Instead, this phosphorylation triggers the sec-nd step of signal termination, by marking activated receptors forinding of arrestin proteins to the phospho-receptor [36,62–64].

The GRK family consists of seven members that are catego-ized into three subfamilies. GRK1 subfamily consists of GRK1rhodopsin kinase) and GRK7 (cone kinase); the GRK2 subfamily isomposed of GRK2 and GRK3; and the GRK4 subfamily is formed byRK4, GRK5 and GRK6 [45,63]. The GRK1 kinases are predominately

ocalized to retinal tissue and influence visual acquisition by regu-ating visual GPCR. The GRK2 subfamily, in contrast, is ubiquitouslyxpressed and possesses a unique ��-subunit binding domain. The�-subunit binding domain of this subfamily permits translocationf these GRKs to the inner leaflet of the plasma membrane uponnteraction with the ��-subunit released by a receptor-activated

protein. In the GRK4 subfamily, GRK5 and GRK6 are expressedn many tissues, while GRK4 expression is primarily localized tohe testes. Within the GRK family, GRKs 2, 3, 5, and 6 are the

ost widely expressed isoforms that are responsible for the bulkf receptor phosphorylation throughout the body.

A large mismatch exists between the number of GPCRs and theumber of GRKs expressed within an organism [36,62–64]. Inheritithin this mismatch, GRKs are relatively promiscuous in recog-izing receptor. While there appear to be some GRK preferencesmong receptor types, specificity is largely driven by the contextf GPCR activation and by relative GRK concentrations in a cell ofnterest, rather than by recognition of any consensus phosphory-ation site [65]. GRKs exhibit some preference for phosphorylationites on receptors, but many studies have shown that GRKs will findther sites to phosphorylate on a receptor if their preferred sites arenavailable, such as after receptor mutagenesis.

.3. Arrestin signal termination

Arrestins constitute a scaffold protein family that initiate theecond part of GPCR signal termination [38,45]. Arrestins recognizective GPCRs and their binding affinity is augmented by GRK medi-ted phosphorylation. Arrestins have varying affinities for differenthosphorylation patterns. Highly phosphorylated serine/threoninelusters induce stronger arrestin-GPCR binding. The arrestin fam-ly consists of four proteins, arrestin1-4. Arrestin1 and arrestin4re unique for their involvement in visual processing. These pro-eins are expressed in the rods and cones of retinal tissue, whereinhey bind to phospho-rhodopsin and phospho-iodopsin to regu-ate GPCR mediated visual perception [63,66,67]. Unlike the visualrrestins, Arrestin2 (aka �arrestin1) and arrestin3 (aka �arrestin2)re expressed in most tissue types [63].

Arrestins promote GPCR signal termination by two distinct, butomplementary mechanisms: receptor desensitization and recep-or sequestration (Fig. 2). These scaffold proteins mediate receptoresensitization by directly inhibiting GPCR-G protein coupling


45]. Mechanistically, arrestin-bound GPCRs are sterically blockedrom interacting with their cognate G protein [38]. This occurslmost immediately after receptor activation, since G proteins,RKs, and arrestins compete for binding to the receptor in its

PRESSmental Biology xxx (2016) xxx–xxx 5

active state. Thus the relative abundance of these three classesof proteins in a cell determine much of the kinetics of receptorsignaling. Though GRK-mediated receptor phosphorylation maypartially accomplish G protein decoupling, the binding of arrestinsto a GRK-phosphorylated GPCR significantly augments this signalblockage mechanism [35,38,46]. Efficient arrestin binding resultsin homologous desensitization, or the turning off of those specificreceptors that have been recently stimulated.

Arrestin-mediated receptor trafficking from the cell surface, orsequestration, occurs minutes to hours after agonist stimulation.Arrestins act as adaptors to associate bound receptors with cellu-lar endocytic machinery proteins to mediate GPCR internalizationinto vesicles as specific cargo. Arrestin binding to phosphorylatedGPCRs promotes arrestin interaction of adaptin and clathrin, twoproteins responsible for mediating receptor internalization [68,69].GPCR internalization sequesters the activated receptors from fur-ther access to agonists outside the cell and effectively reduces thenumber of receptors present on the cell surface for ligand stimula-tion.

After mediating GPCR internalization, arrestins may either dis-sociate or remain bound to a receptor as it begins to transit throughintracellular vesicular compartments, depending on arrestin affin-ity for the phospho-receptor [70]. Early dissociation of arrestin froma weak-binding GPCR keeps the receptor on a quick itinerary lead-ing to receptor recycling, wherein the endocytosed GPCR quicklyreturns to the cell surface to engage in further ligand binding(Fig. 2). Failure of the arrestin-GPCR complex to dissociate earlymarks that receptor for further processing through intracellu-lar compartments that result in either receptor degradation ordelayed receptor recycling [22,49]. Among other factors, the con-formational state of the receptor and the pattern of GRK-mediatedphosphorylation collectively influence arrestin-GPCR bindingdynamics .

GRK/arrestin signal regulation is stoichiometric, with onearrestin molecule able to bind to and regulate only one GPCR. Lowreceptor ligand occupancy can induce full G protein signaling for anextended period with low desensitization, since a small number ofreceptors can activate many G proteins through amplification, butalso leads to only a few receptors at a time being removed from afunctional state on the cell surface. The endocrine system demon-strates this phenomenon, where agonists released into the systemiccirculation are highly diluted within the vasculature that carriesthem to their target tissues where they can have sustained effects.In contrast, high receptor ligand occupancy will induce an intenseburst of G protein activity, followed by profound GRK/arrestinmediated desensitization. This type of regulation is most evident atneuronal synapses, where a neurotransmitter agonist is released inlarge quantities into synapses, and signaling occurs in bursts ratherthan constantly.

4.4. Arrestin GPCR feedback regulation

Arrestins additionally regulate GPCR activity through non-adapter based feedback regulatory mechanisms. The �2AR systemis regulated by arrestin through feedback regulation. �2AR acti-vation yields an increase in PKA activity secondary to cAMPproduction. Phosphodiesterase (PDE) opposes this activity bydegrading cAMP. Upon �2AR activation, arrestin scaffolds withPDE4D and promotes its translocation to the plasma membrane[71]. PDE4D translocation and activation negatively regulates the�2AR cascade by degrading the substrate necessary for PKA acti-vation. Interestingly, observations demonstrate that these events


additionally inhibit PKA mediated phosphorylation of active �2AR[71]. Hindrance of PKA-mediated �2AR phosphorylation inhibitsG protein switching [33,61]. Arrestins additionally partake in Gq-coupled M1 muscarinic receptor feedback regulation [72]. DAG



6 J.B. Black et al. / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

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Fig. 2. 7TMR/GPCR signal regulation. (A) Receptor desensitization. GRKs phosphorylate the C-terminal of activated GPCRs. Arrestin binds to the phosphorylated receptorthat is then sterically blocked from interacting with cognate G protein. (B) Receptor sequestration. Arrestin serve as an adaptor and recruit endocytic machinery proteins tom vatedt lure ot

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ediate GPCR internalization into vesicles. GPCR internalization sequesters the actihe receptor in vesicle. Early dissociation of arrestin promotes receptor recycling. Faihat results in either receptor degradation or delayed receptor recycling.

nd IP3 are formed upon Gq stimulation. Diacylglycerol kinasesDGKs) terminate Gq signaling by phosphorylating the second


essenger DAG. Similar to PDE4D interactions in the �2AR-Gs sys-em, arrestins scaffold with and activate DGKs to degrade DAG.ffectively, arrestin promotes Gq negative feedback regulation byctivation removal of the second messenger DAG (Fig. 3).

receptors from further stimulation. (C) Arrestin may dissociate or remain bound tof the arrestin-GPCR complex to dissociate marks that receptor for further processing

5. GRK and arrestin-mediated signaling


Arrestins and GRKs also function in GPCR signal termination-independent processes. They serve as molecular scaffolds that canalter the activity of partners including protein kinases, transcrip-tion factors, and other cellular signal modulators. GRKs can regulate



J.B. Black et al. / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx 7

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Fig. 3. GPCR feedback regulation. (A) G protein switching. Upon initial stimulation, �2AR activates the Gs protein to increase adenylate cyclase signaling and PKA activityto propagate the cellular response. PKA initiates G protein switching by phosphorylating site-specific residues on �2AR. PKA phosphorylation of �2AR alters the receptorcoupling from Gs to Gi. Activated Gi yields a decrease in cAMP production and PKA activity. (B) Arrestins partake in Gq-coupled M1 muscarinic receptor feedback regulation.A a meG DE4Da neces

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rrestin scaffolds with diacylglycerol kinases (DGKs) and recruits them to the plasmq negative feedback regulation. (C) Upon �2AR activation, arrestin scaffolds with Pctivation negatively regulates the �2AR-cAMP cascade by degrading the substrate

he activity of many cytoplasmic and nuclear non-GPCR substrateshrough both scaffold- and kinase activity-dependent functions.

.1. Non-canonical GRK activity

Accumulating evidence highlights a non-canonical GRK func-ion in regulating cellular processes independent of GPCR signalermination. Non-canonical GRK activity extends to regulating non-PCR substrates, cytoskeletal dynamics, protein catalytic activitynd other diverse functions. Non-GPCR GRK substrates are doc-mented in several cellular compartments, including the plasmaembrane, cytoplasm, and nucleus. This kinase family regulates

on-GPCR substrates in both kinase-dependent and -independentashion [36,62,63].

GRK2 and GRK3 have two distinct receptor-independent func-ions in regulating G proteins. As noted previously, GRK2 family

embers bind to G protein ��-subunits released by receptor-ctivated G proteins [36,63]. Since ��-subunits are plasmaembrane anchored, this recruits GRK2 to the plasma membrane

n the vicinity of activated receptors. Although ��-subunit bind-ng does not directly alter GRK2 kinase catalytic activity, GRK2an in fact be considered a ��-subunit effector because this effi-ient localization greatly enhances the effectiveness of the kinase.n addition, GRK2 competes for G protein ��-subunits with other�-stimulated effectors, and this domain has been used widely as

��-subunit scavenger to assess G� versus G�� functions [20,73].isease states that increase or decrease GRK2 levels thus alter G��

ignaling tone. The second G protein function is that GRK2 and GRK3lso bind to activated Gq �-subunits through their RGS-like domain.gain, activated Gq does not appear to alter GRK2 function, butRK2 does compete with PLC-� and other effectors for activatedq subunits.

GRK-mediated phosphorylation regulates non-GPCR receptorctivation. For instance, the platelet-derived growth factor receptor

(PDGFR�) is a single-transmembrane span tyrosine kinase recep-or responsible for mediating cellular proliferation and survivalignals upon ligand stimulation. GRK2 negatively regulates PDGFR�inase activity by phosphorylating specific serine residues, thusecreasing PDGFR�-mediated phosphorylation of its substrates


74,75]. Similarly GRK5/6 positively regulate the tyrosine kinaseeceptor, insulin-like growth factor-1 receptor (IGF-1R). Indeed,GF1-mediated ERK and AKT activation is diminished in cells whenither GRK5/6 expression is decreased [76].

mbrane. These events activate DGKs to degrade DAG, promoting arrestin-mediated and promotes its translocation to the plasma membrane. PDE4D translocation andsary for PKA activation.

GRKs regulate cytoskeletal dynamics through GPCR-dependentand -independent mechanisms. The ezrin–radixin–moesin (ERM)protein family constitutes three GRK-regulated proteins that medi-ate cytoskeletal rearrangement [77,78]. ERM proteins localizeto the cytoplasm and promote cytoskeletal rearrangement byfacilitating crosstalk between plasma membrane-bound proteinsand F-actin [79]. GRK-mediated phosphorylation on specific ERMresidues conformationally activates ERM to yield F-actin poly-merization. GRK mediated ERM activation promotes cytoskeletonremodeling and cell adhesions [62]. Interestingly, GRK2 medi-ates muscarinic M1 receptor membrane ruffling through ezrinphosphorylation [62,78]. Furthermore, GRK5 mediated moesinphosphorylation promotes cellular invasion and migration [77].Additionally, GRKs influence microtubule (MT) processing, whichfunctions in cytoskeletal polarization and migration [80]. MTdynamics are regulated by post-translational modifications, suchas acetylation and phosphorylation [81]. Indeed, GRKs phosphory-late tublin, the basic monomer of MT [82]. GRKs phosphorylate andactivate histone deacetylase 6 (HDAC6) to deacetylate �-tubulin,an MT subunit. GRK mediated HDAC6 deacetylation promotes MTpolymerization [80].

This kinase family additionally regulates nuclear proteinsinvolved in diverse functions, such as gene transcription andepigenetics. Exeplar transcription factor NF-�B undergoes GRK5-mediated regulation. I�B family members negatively regulateNF-�B by binding and sequestering NF-�B in the cytoplasm. I�Bkinase (IKK) phosphorylation of I�B family members promotesNF-�B dissociation and subsequent I�B family member degrada-tion. GRK5 additionally mediates I�B family member degradationand NF-�B activation by phosphorylating this protein family uponTNF� stimulation [83]. Interestingly, additional reports argue thatGRK5 can oppose NF-�B activity through mediating I�B� nuclearaccumulation [84]. Furthermore, GRK5 augments nuclear factorof activated T cells (NFAT) transcriptional activity in an apparentkinase independent manner, potentially by some molecular scaf-fold activity [85].

5.2. Arrestin-mediated signaling


Arrestin-mediated GPCR signal termination constitutes theirdefining function. Available evidence, however, highlights thatarrestins additionally mediate signal transduction, cellular organi-zation, cytoskeletal dynamics, and other cellular processes through



8 J.B. Black et al. / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

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eceptor conformational changes that can result in arrestin signaling activation. Arellular processes. (B) Arrestins regulate protein spatiotemporal distribution. GPCRromotes nuclear translocation of ERK and subsequent activation of downstream

ith GPCR-bound arrestin. Vesicle-bound ERK is sequestered in the cytoplasm and

olecular scaffold activity [45]. Molecular scaffolds regulate theubcellular spatiotemporal distribution of proteins, promote com-lex formation by bridging interacting components, and reduceff target effects by increasing reaction fidelity. Proteomic screensdentified novel arrestin interactors in diverse pathways, suchs signal transduction, motor processing, metabolomics, andytoskeletal arrangement [22]. This scaffold ability increasesechanistic impact of arrestins on cell physiology in both GPCR-

ependent and -independent states [22,38].Arrestins extend the GPCR signaling pathway beyond G proteins


y scaffolding with kinases. GPCR/arrestin-mediated scaffoldingccurs secondary to GPCR agonist activation [86]. For instance,rrestin-bound GPCR promotes tyrosine kinase Src recruitmentFig. 4). Arrestin-bound GPCR enhances the catalytic activity

mediated signaling is independent of G protein coupling and can regulate diverselation promotes ERK activation via G protein mediated mechanisms. This pathwayription factors. Arrestin mediates an alternative pathway wherein ERK complexestes a separate set of effectors.

of Src to further diversify cellular effects, including promot-ing further GPCR signaling. Src-bound arrestin potentiates GPCRsignaling by targeting GRKs for degradation, thus inhibiting theinitial step of GPCR signal termination [70]. Furthermore, the Src-arrestin complex extends GPCR signaling pathways by promoting Gprotein-independent signaling. This complex trans-activates otherpathways, such as the pro-mitogenic EGF receptor that can activateERK to initiate pro-mitogenic transcriptional activity [23,87].

Arrestins exert a contextual influence on kinases. Their scaf-folding ability can either directly or indirectly alter kinase


activity. Src-independent arrestin ERK activation demonstratesdirect arrestin mediated regulation. Arrestin2 and 3 each complexwith ERK independent of Src [22]. However, Arrestin2 and 3 main-tain opposing functions. Arrestin2 inhibits ERK activation, while


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rrestin3 augments the kinase activity [22]. In contrast, arrestinndirectly regulates the transcription factor NF-�B by directlyltering the stability of a regulatory scaffold protein. Arrestin indi-ectly promotes NF-�B inhibition by stabilizing I�B�. Arrestin-I�B�caffolding prevents IKK mediated phosphorylation and subse-uent NF-�B release. Consequently, arrestin indirectly regulatesF-�B by stabilizing the negative regulator protein responsible for

ts cytoplasmic localization [88] (Fig. 4).Scaffold proteins regulate the spatiotemporal distribution of

ellular components ERK activation post GPCR activation sup-orts this conclusion. GPCR stimulation promotes ERK activationia G protein mediated mechanisms. Activation by this pathwayesults in nuclear translocation of ERK and subsequent activation ofownstream transcription factors. Arrestin mediates an alternativeathway: ERK can form a complex with GPCR-bound arrestin via a

protein-independent mechanism. ERK recruitment to the plasmaembrane results in its presence in clathrin-coated pits togetherith GPCR and arrestin. Vesicle-found ERK is sequestered in the

ytoplasm where it activates a separate set of effectors distinct fromhe subset of G protein-activated ERK substrates [22,89,90].

.3. GPCR biased signaling

GPCR ligand activation induces receptor conformationalhanges, which stimulate downstream effector molecules. The Grotein and arrestin signal pathways present two exclusive effec-or molecules influenced by GPCR structural dynamics. ActivePCR conformations are dynamic and exist in several structural

orms, with partially non-redundant functions. Indeed, certainPCR conformations specifically promote either a G protein orrrestin signaling pathway [91]. Ligand-receptor dynamics reflecthis conformational complexity. Balanced ligands permit all poten-ial active GPCR conformations, while biased ligands only permitPCR to assume a subset of potential active structures [34,92,93].

n contrast to balanced ligands, biased ligands induce conforma-ions that permit the specific activation of either G protein orrrestin based signaling [34,92,93]. For example, carvedilol, an FDApproved beta-blocker, demonstrates biased activity in the car-iovascular system. This therapeutic induces GPCR conformationshat selectively activate arrestin pathway, without stimulating Grotein activity [94].

. Perspective

GPCR-mediated signal transduction presents an intricateechanism for controlling cellular activities and facilitating

ommunication. Diverse processes ranging from cardiovascularemodeling to endocrine signaling utilize GPCR activity for elicitingellular responses. Pharmacotherapeutics target GPCR pathwaysor clinical interventions due to their applicability in controllinglinically-relevant pathways. Indeed, over 50% of pharmacologicnterventions target GPCRs. However, GPCR’s intricate and diver-ified mechanisms of activity contribute toward unwanted sideffects. Consequently, fundamental advances in GPCR biologyirectly influence novel pharmacologic development void of dele-erious effects. Indeed, gained knowledge in GPCR structure andignal transduction augments the opportunity for smart drugevelopment seeking to produce receptor signal-specific ther-pies without off-target effects. Identifying GPCR conformationtates that selectively activate either arrestin or G protein signalingetworks presents an additional control to prevent drug deleteri-


us side effects. While GPCRs present a heavily targeted family,ess than 10% of GPCRs currently are targeted for interventions.his offers the opportunity to identify and target new GPCRs. Over-ll, the advancement of GPCR research presents profound clinical

[

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PRESSmental Biology xxx (2016) xxx–xxx 9

applications inherit within their influence of cellular dynamics.Recognizing GPCR regulatory feed-back loops expands opportuni-ties to harness specific signals for therapeutic interventions.

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