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Cell Communication (GPCRs) (Chapter 16, part 1)

General principles of cell signalling GPCRs and second messengers

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Signal transduction

• Cell signalling requires a signal molecule (from a signalling cell) and a receptor protein (on a target cell).

• The receptor recognizes the signal (= signal reception).

• The cell responds specifically to the signal molecule (= signal transduction).

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Different forms of cell signalling

Hormones: Steroid hormones

Peptide hormones

Amino acid derivatives

Local mediators: Growth factors (proteins)

Histamine

NO (gas)

Neuro-transmitters: Glutamate

GABA

Acetylcholine

Membrane proteins: Delta

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Endocrine vs. synaptic signalling

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Autocrine signalling

• Signal molecule binds back to a receptor on the same cell type. • This receptor is called an autoreceptor. It is sensitive to the signalling molecule

released by the cell in whose membrane the autoreceptor sits. (feedback loop, threshold sensor).

• Autocrine  signalling  can  coordinate  groups  of  identical  cells  (“community effect”),  e.g. cell fate decisions during development.

• Amplified autocrine cell signalling by factors stimulating cell survival or proliferation is one of the hallmarks of cancer.

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Contact-dependent signalling

Delta and Notch are both transmembrane proteins controlling embryonic development and cell differentiation (e.g.neurons and glial cells)

”lateral  inhibition”

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The same signal molecule (acetylcholine) can induce different responses in different target cells

• In heart muscle and sailvary glands, acetylcholine binds to similar receptors (muscarinic AchR, a GPCR), but these activate different responses (decreased contraction, increased secretion)

• In skeletal muscle, a different acetylcholine receptor (nicotinic AchR, an ion channel) enables these cells to react differently (increased contraction)

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Animal cells depend on multiple extracellular signals

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Fast and slow responses to extracellular signals

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Signal molecules bind either to cell-surface receptors or to intracellular proteins

Some hormones (hydrophobic signal molecules) can cross the plasma membrane and bind to intracellular receptors

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The steroid hormone cortisol acts by activating a gene regulatory protein

(a intracellular/nuclear receptor protein)

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Nitric oxide (NO) triggers smooth muscle relaxation

• NO acts as local mediator in many tissues • Endothelial cells release NO (made from arginine by NOS) • NO activates soluble guanylyl cyclase in muscle cells • Increased cGMP induces muscle cell relaxation

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Extracellular signals alter the activity of a variety of cell proteins to change the behavior of the cell

Signalling cascade

• Amplification

• Distribution

• Modulation

Response

Signal reception

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Cellular signaling cascades can follow a complex path

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Many intracellular signalling proteins act as molecular switches

Protein phosphorylation can only occur at serine (S), threonine (T) or tyrosine (Y) residues

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The three main classes of cell-surface receptors

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G protein-coupled receptors (GPCRs, 7TM receptors)

trimeric G-protein GPCR

ligand

GPCRs are excellent drug targets

• In humans, about 40% of all medical drugs act on GPCRs.

• GPCRs play a central role in human physiology.

• GPCRs are feasible targets for the development of non-peptide agonists and antagonists

• In humans, there are about 800 genes coding for GPCRs (about 400 of them are olfactory receptors).

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2012 nobel prize in Chemistry for GPCR research

• R. Lefkowitz cloned the first GPCR gene in 1986 (the β2-adrenergic receptor), worked on signal transduction and regulation of adrenergic receptors, his lab discovered GPCR kinases (GRKs) and E-arrestins

• B. Kobilka worked on GPCR structure, his lab solved the 3D-structures of the β2-adrenergic receptor in inactive, active and G protein-bound states

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Proteins of the GPCR signaling cascade

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Family A: Rhodpsin/E2 adrenergic receptor-like

Gether U. (2000) Endocrine Reviews 21(1): 90–113

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Family B: Glucagon/VIP/Calcitonin receptor-like

Gether U. (2000) Endocrine Reviews 21(1): 90–113

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Family C: Metabotropic glutamate receptor-like

Gether U. (2000) Endocrine Reviews 21(1): 90–113

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The G-protein disassembles into two signalling proteins when activated

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The D-subunit switches itself off by hydrolyzing its bound GTP

•The activated D subunit can activate a target protein

•The activated EJ complex can activate another target protein

•After a few seconds the intrinsic GTPase activity of the D subunit hydrolyzes GTP to GDP

•This inactivates the D subunit and the trimeric complex is reformed, thereby also inactivating the EJ�complex

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heart contracts less frequently

Signalling of the activated EJ complex: an acetylcholine receptor in heart muscle

cells opens a K+ channel in the PM

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3 main classes of D-subunits: Gs, Gi, Gq

• Cholera toxin: covalent modification of Gs (remains active)

• Pertussis toxin: covalent modification of Gi (remains inactive)

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Most G proteins activate the synthesis of intracellular messenger molecules

“second  messengers”

1. cAMP pathway: cAMP 2. Inositol phospholipid pathway: IP3 and DAG, Ca2+

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G proteins regulate the activity of adenylyl cylase

cAMP activates the A-kinase (cAMP-dependent protein kinase, PKA)

A-kinase phosphorylates serines or threonines on certain intracellular proteins, thus altering their activity.

cAMP is synthesized by adenylyl cyclase and degraded by cyclic AMP phosphodiesterase

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The cAMP concentration rises rapidly in response to an extracellular signal

Blue: low cAMP concentration

Red: high cAMP concentration

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Enzyme activation by cAMP

PKA: cAMP-dependent protein kinase

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Activation of gene transcription by cAMP

PKA: cAMP-dependent protein kinase

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Phospholipase C activates the inositol phospholipid pathway

Second messengers:

• IP3 -> Ca2+

• DAG

The activated PKC (protein kinase C, Ca2+-dependent) can further phosphorylate many different intracellular proteins

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Ca2+-calmodulin

binding of 4 calcium ions induces a conformational change

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Ways to keep a low intracellular free Ca2+ concentration

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• In the human olfactory epithelium there are about 10 million olfactory sensory neurons

• Humans have about 350 different olfactory receptors

Smell depends on GPCRs that regulate ion channels

Olfactory GPCRs in cell membrane

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Olfactory transduction in the cilia of olfactory sensory neurons

• The odorant binds a specific odorant receptor (OR) • The activated OR activates Golf • Golf activates adenylyl cyclase to increased cAMP • cAMP opens cAMP-gated Na+-channels (olfactory CNG

channels) • influx of Na+ depolarizes the olfactory receptor neuron

and initiates an action potential

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Cyclic nucleotide gated (CNG)-channels • cAMP-gated channels are found in

olfactory sensory neurons

• cGMP-gated channels are located in the outer segment of vertebrate photoreceptor cells

• CNG channels are tetramers The different CNG channel subunits adopt similar topologies with 6 transmembranal segments (S1-S6) and a pore forming region between S5 and S6.

• The amino (N) terminus and the carboxy (C) terminus, containing the cNMP-binding site, are located intracellularly.

• When cyclic nucleotides bind, conforma-tional rearrangements lead to the opening of the pore.

• The channels are permeable for Na+ (-> de-polarization), K+ and Ca2+ ions.

cAMP- or cGMP-binding site

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Richard Axel Linda B. Buck

1/2 of the prize 1/2 of the prize

USA USA

Columbia University New York, NY, USA; Howard Hughes Medical Institute

Fred Hutchinson Cancer Research Center Seattle, WA, USA; Howard Hughes Medical Institute

b. 1946 b. 1947

The Nobel Prize in Physiology or Medicine 2004 "for their discoveries of odorant receptors and the organization of the olfactory system"

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Vision depends on GPCRs: Rhodopsin, a combination of 11-cis-retinal

(yellow) and the protein opsin

Extracellular signal: light

Receptor: opsin

Transducer (G protein): transducin

Primary effector: phosphodiesterase

Intracellular signaling molecule (second messenger): cGMP ( )

opsin (a GPCR)

11-cis-retinal (chromophore)

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Vision depends on photoreceptors in the eye • light activates the GPCR

rhodopsin

• rhodopsin activates the G protein transducin

• transducin activates a phosphodiesterase (PDE) to hydrolyze cGMP

• cGMP normally keeps cGMP-gated Na+-channels open.

• the drop in cGMP closes more of the cGMP-gated Na+-channels

• the photoreceptor cell hyperpolarizes

• less glutamate is released to the bipolar cell in light

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CNG channels in phototransduction

• In darkness, open CNG channels in the cone outer segment conduct a depolarizing current carried by Na+ and Ca2+ ions.

• Upon illumination, CNG channels close and the cone hyperpolarizes.

low cGMP level (active PDE)

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Amplification in the photoreceptor cascade

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Ways how a cell can become desensitized to a signal molecule (adaptation)

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Adaptation: Desensitization of GPCRs by GRKs and arrestins

• A GPCR kinase (GRK) phosphorylates the activated receptor

• Arrestin binds to the phosphorylated receptor

• This prevents the receptor from binding to its G protein

• It can also direct its endocytose (arrestin as adaptor protein)

• Arrestin can also redirect the receptor complex to other signalling pathways or act as scaffolding protein

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GPCR trafficking - GRKs and arrestins

downregulation

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Crystallization of GPCRs

Problems:

1. GPCRs are hydrophobic membrane proteins

2. Usually low expression levels

3. Conformational instability

Solutions:

1. A source that contains the GPCR in high amounts.

2. Replacement of the intracellular loop 3 (ICL3) with a well-folded, polar protein (T4 lysozyme)

3. Binding of a monoclonal antibody (Fab5) to stabilize the ICL3

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� >50 insect genomes have been sequenced � many  more  are  in  the  pipeline  (“i5k”)  

Our aim: - to advance our understanding of insects - to contribute to crop protection - to fight diseases transmitted by insects

Our focus: - insect endocrinomics - GPCRs and neuropeptides - ligand/receptor matching (deorphanization)

reproduction

development

feeding

behaviour

30%

70%

(NIH supported consortia) TCTGTACAAGATCCGTGGGACGAATGCGATCCACCAGCAGAGCCATCAATTCGATACCAAATCCCTTATTATAGCCCATCGTATTTATCAGCCAAGGAATATTTGAATACTTGGCATCGTTCTGAATGTTTTGGACTAGCTGAATAACTGCTCGGGCATACTGCTCTGCGCACAAGACAATGTTGGTGTGGCCCACGACAATGGCATGTTCCGGCTGCCTGTTGTACAGGAAGCCCGGTCCCAGAAGGGGCTCATCAATTACGGTGCAAGAAATAGTCTGGGGTACAAATATTTCCGGCTGACCAATGTCCAGGTCGATGAGCAGCATGGATGGGAATTGACCCAGATTTCGGTTGATGAGGTAACGCAATAAACTAGACTTTCCGACGCCCTTACCACCGGCTAC

Fruit fly

Mosquitoes

Honey bee

Flour beetle

Silk worm

Parasitic wasp

Blood-sucking bug

Body louse

Pea aphid

Center for Functional and Comparative Insect Genomics (Biol. Inst., University of Copenhagen)

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Reverse Pharmacology Strategy

ligand library

”orphan”  receptors  expressed  in  cells

similar to HTS (high throuput screening) in drug discovery

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Our bioassay using G16 in CHO cells: Ca2+- induced bioluminescence

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CHO cells expressing the proctolin receptor

Control cells without proctolin receptor

Cells expressing the proctolin receptor

� proctolin

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Cloning and matching of (new) receptors and (new) ligands in various insects

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Receptor localization

In vivo function

Correlation with physiology

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Correlation of hormonal systems with insect physiology and lifestyle

Functional in vivo studies by gene knock-down

Identification of new functions by localization of neuropeptides and receptors

Systematic comparison of the hormonal set-up of insects: endocrinomics

Bachelor or master projects availabe:

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