signal reception: g protein-coupled receptors

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Signal Reception: G Protein-Coupled Receptors

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Signal Reception: G Protein-Coupled Receptors. Neurotransmitter receptors. Ligand – gated channels: Nicotinic acetylcholine receptor NMDA-type glutamate receptor Glycine receptor GABA A receptor Serotonin receptor (5-HT 3 ) G protein-coupled receptors: - PowerPoint PPT Presentation

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Page 1: Signal Reception: G Protein-Coupled Receptors

Signal Reception: G Protein-Coupled Receptors

Page 2: Signal Reception: G Protein-Coupled Receptors

Neurotransmitter receptors

Ligand – gated channels:• Nicotinic acetylcholine receptor• NMDA-type glutamate receptor• Glycine receptor• GABAA receptor• Serotonin receptor (5-HT3)

G protein-coupled receptors:• Muscarinic acetylcholine receptor (several types)• Catecholamine receptors • Histamine receptors (H1, H2)• 5-HT receptors other than 5-HT3

• GABAB receptors• ‘Metabotropic’ glutamate receptors• Peptide receptors (Endorphin, cholecystokinin..)

Page 3: Signal Reception: G Protein-Coupled Receptors

The G Protein-Coupled Receptor (GPCR) Superfamily

• Largest known receptor family – Constitutes > 1% of the human genome.

• Comprises receptors for a diverse array of molecules: neurotransmitters, odorants, lipids, neuropeptides, large glycoprotein hormones.

• Odorant receptor family alone contains hundreds of genes.

• Mammalian GPCRs: nearly 300 different kinds – grouped into 3 main subfamilies:

Page 4: Signal Reception: G Protein-Coupled Receptors

Three Main Mammalian GPCR Subfamilies

• Rhodopsin-like group – includes most of the GPCRs.

• Glucagon-like group.• Metabotropic glutamate (mGlu) and GABAB

receptor family.

Page 5: Signal Reception: G Protein-Coupled Receptors

Three Main Mammalian GPCR Subfamilies (cont’d)

• Grouped according to > 20 % sequence homology.• Databases for the classification of receptors into

subfamilies, phylogenetic trees, chromosome localization, ligand binding constants and receptor mutations can be found at www.gpcr.org/7tm

Page 6: Signal Reception: G Protein-Coupled Receptors

Almost all Receptors Comprise a Number of Subtypes

• Dopamine receptors - 5 subtypes• 5-HT receptors – 13 subtypes• mGlu receptors - 8 subtypes• Acetylcholine receptors – 5 subtypes• Identified by their pharmacological and functional

characteristics, rather than by strict sequence homology:- Some receptors for the same ligand show remarkably little homology (e.g., histamine H3 and H4 have the lowest recorded homology (~ 20 %) to other histamine receptors H1 and H2).

Page 7: Signal Reception: G Protein-Coupled Receptors

• Each GPCR family contains some orphan receptors, which have been identified as members of the GPCR superfamily by homology cloning but whose activating ligand is unknown.

• But high throughput screening has recently added to the advances in being able to identify the ligand.

Page 8: Signal Reception: G Protein-Coupled Receptors
Page 9: Signal Reception: G Protein-Coupled Receptors

Originally published in Science Express on 25 October 2007.Paper version: Science 23 November 2007: Vol. 318. no. 5854, pp. 1258 - 1265.

High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor

Vadim Cherezov, Daniel M. Rosenbaum, Michael A. Hanson, Søren G. F. Rasmussen, Foon Sun Thian, Tong Sun Kobilka, Hee-Jung Choi, Peter Kuhn, William I. Weis, Brian K. Kobilka,Raymond C. Stevens

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β2-adrenergic receptor–T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein–coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

Page 10: Signal Reception: G Protein-Coupled Receptors

Types of G Proteins and their 2nd Messenger Pathways

Page 11: Signal Reception: G Protein-Coupled Receptors

α, β, γ Subunitsα Subunit (23 isoforms): contains the GTP/GDP binding site

is responsible for identity. β (5 isoforms) and γ (12 isoforms): are identical or very

similar, interchangeable in vitro; most of them are ubiquitously expressed; membrane anchored through prenylation of Gβ.

Golf: expressed in olfactory bulb, coupled to PLCβ.GT (transducin): is coupled to cGMP phosphodiesterase and is

expressed in the rod cells of the retina (these cells are Inactivated by light!!): hν hits rhodopsin -> opsin is activated -> facilitates GTP loading of GT -> activates cGMP phosphodiesterase -> cGMP (keeps Na+ and Ca2+ channels open to cause depol -> nt release) -> converted to 5’GMP (inactive => channels closed => membrane polarization => no nt released).

Page 12: Signal Reception: G Protein-Coupled Receptors

Receptor Family 1 – Rhodopsin Family

TM2

TM3

TM1

TN4

TM5

TM6

TM7

NY Y

Extracellular

IntracellularDRY

CC

C

C C

Lipid Bilayer

Page 13: Signal Reception: G Protein-Coupled Receptors

Receptor-Ligand Interactions:Rhodopsin-like Family

32

1

7

6

4

5N

F

D

+N

HO

OHOH

S

S

Page 14: Signal Reception: G Protein-Coupled Receptors

Receptor Family 2 – Glucagon-like Family

• Structurally similar to that of Family 1, except that they have a much larger N-terminal domain, which contains multiple potential S-S bridges.

• Most of the ligands binding to these receptors are peptides or glycoprotein hormones:

- 30-40 residues is typical.- likely to interact with the receptor over large surface areas.

Page 15: Signal Reception: G Protein-Coupled Receptors

Receptor Family 2 – Glucagon-like Family

TM2

TM3

TM1

TN4

TM5

TM6

TM7

Y Y

Extracellular

IntracellularDRY

CC

C

C C

Lipid Bilayer

N

Page 16: Signal Reception: G Protein-Coupled Receptors

Receptor Family 3 – mGluR/GABAB Family• Extremely large extracellular N-terminal ligand binding

domain.• Highly conserved 3rd short intracellular loop.• Shares only ~ 12 % sequence homology with that of Family 1,

but the overall transmembrane topology is similar.• Forms dimers:

- GABAB receptor forms heterodimers between GABABR1 and GABABR2 through coiled coil regions in the C-terminal tails.

- This dimerization is required for efficient cell surface expression and signalling.

- Metabotropic glutamate receptors dimerization is stabelized by disulfide bonds in the N-terminal extracellular domain.

Page 17: Signal Reception: G Protein-Coupled Receptors
Page 18: Signal Reception: G Protein-Coupled Receptors
Page 19: Signal Reception: G Protein-Coupled Receptors

Common Experimental Tools used to Study GPCRs

Page 20: Signal Reception: G Protein-Coupled Receptors

D D

ααβ

αγ

GDP

Drug bindingand G protein

activationααβ

αγ

GTP

D

Dissociation ofreceptor-G protein

complex

αα

βα

γ GTP

Pi

αα

GDP

Reformation ofreceptor G protein

complex

Inactivation of Gα through intrinsic

GTPase activity

The G Protein Cycle

Page 21: Signal Reception: G Protein-Coupled Receptors

Receptor-G protein InteractionsHow are receptor-G protein interactions measured?

• Ligand-binding assays:

High-affinity Low- affinityRG(GDP)

GDP GTPγS

R + G(GTP-δ-S)

Without GTP, both high- and low-affinity states are measured.With GTP and Mg2+, only low-affinity state is measured, becauseAgonist binding rapidly induces change from high- to low-affinity.

Page 22: Signal Reception: G Protein-Coupled Receptors

Receptor-G protein Interactions Structural features of receptors involved in G protein activation

How does agonist binding cause receptor conformational change?

• Agonists vary in their binding affinity for the GPCR = drug-receptor interaction.

• How well the drug causes a conformational change in the receptor to activate G proteins = efficacy.

• There are multiple agonists (partial, full) with different binding affinities.

Page 23: Signal Reception: G Protein-Coupled Receptors

Receptor-G protein Interactions Structural features of receptors involved in

G protein activation

• Mechanism of conformational change highly conserved.• Constraining intermolecular interactions that keep receptors

preferentially silent in the absence of agonist: such as between TM5 & TM6 and between TM3 & TM7.

• E.g., ‘DRY’ motif in TM3 (earlier).• Upon receptor activation, the arg is protonated adjacent

residues move tilting the TM helix incr exposes previously hidden sequences, which interact with G protein.

• Much evidence for preceding.• However, the exact aa sequence responsible for this has been

difficult to pinpoint.

Page 24: Signal Reception: G Protein-Coupled Receptors

Constitutive Activity• Many receptors show constitutive activity even when

expressed at physiol levels (e.g., rat dopamine D1, rat, human hist H2, human dopamine D3, and human 5-HT1A).

• Inverse agonists.• Mutations have been identified that incr the basal activity w/o

affecting the ability of agonists to further activate the receptors.

• These mutations affect stabilizing interactions between helices that hold the receptor in an inactive state and those interfering with these interactions

Page 25: Signal Reception: G Protein-Coupled Receptors

Multiple active conformations – stimulus trafficking• There may be several active conformations, which are

induced by certain drugs = stimulus trafficking.• Different drugs can promote distinct receptor

conformations, which interact with different G proteins resulting in activation of distinct signaling pathways.

• E.g., partial agonists at human 5HT2A and 5HT2C receptors differential stimulation of IP and AA 2nd messenger signaling systems.

Page 26: Signal Reception: G Protein-Coupled Receptors

Cell-type Specific Factors

• Receptor Splice Variants• Levels of receptor expression and signal amplification (see

next slide).- with high receptor density + strong coupling to G protein pathway, the [drug] required to generate 2nd messengers may << [drug] required to occupy a significant fraction of receptors.- This system will show a large amount of signal amplification.- ‘receptor reserve’ = ‘spare receptors’ = ‘strong coupling’.- Signal amplification is fast.- Equilibrium is reached quickly – depends on the rate constants for association and dissociation.

Page 27: Signal Reception: G Protein-Coupled Receptors

% ofMax

100

80

60

40

20

10

Binding

Response

Response

0.01 0.1 1 10 100 1000 10000Drug (nM)

Signal Amplification and Receptor Reserve

KD = 100 nM – goodenough in a stronglycoupled system (left shift).In contrast, the same receptors in this cell may also signal through another, less well coupled pathway with less signal amplification and less receptor reserve.

Page 28: Signal Reception: G Protein-Coupled Receptors

Specificity of receptors for G protein subtypes

Some receptors can show selectivity for a certain α subtype in 1 type of cell, but not in another cell type:

R (muscarinic)

R (somatostatin)

G (α01/β3/γ) E (Ca2+ channels)

G (α02/β1/γ)

Page 29: Signal Reception: G Protein-Coupled Receptors

Restricted localizationGPCRs undergo the same trafficking we have

discussed earlier (Protein trafficking and LGIC slides).

Page 30: Signal Reception: G Protein-Coupled Receptors

Regulation of G protein-coupled receptor function

Desensitization/resensitization – a decrease in responsiveness during continuous drug application or a right-shift in a drug dose-response curve.

After removal of the drug, receptor activity recovers, although the speed and extent of this resensitization can depend on the duration of agonist activation.

Rapid desensitization (sec-min) results from receptor phos, arrestin binding, and receptor internalization.

Long-term desensitization (down-regulation) involve changes in receptor and/or G protein levels, and their mRNA stability and expression.

Long-term changes in [GPCR]s and [accessory proteins]s known to be induced by chronic drug treatment and involved in several pathologies.

Page 31: Signal Reception: G Protein-Coupled Receptors

Phosphorylation2nd messenger kinaseG protein receptor kinase (GRK)Arrestinβ-arrestin binding to phosphorylated GPCR is

required to decrease GTPase activity prior to desensitization.

Receptor trafficking, internalization, and recycling (covered earlier; see Protein trafficking and LGIC slides).

Page 32: Signal Reception: G Protein-Coupled Receptors

Mechanisms of long-term down regulationLong-term (> 1 hr) treatment with agonist induces the loss of

total cellular receptor number in addition to the decr in surface receptor number.

e.g., antidepressants (e.g., fluoxetine) incr [5HT]synapse decr 5HT receptor density.

Receptor endocytosis: C-terminal domain determines whether they enter the recycle pathway or the lysosomal pathway:- 2 distinct motifs: 1. PDZ-domain interats with NHERF in a phos-dependent manner.2. A short sequence that interacts with NSF (N-ethylmaleimide sensitive factor).

Arrestin has also been shown to be important for recycling:e.g., V2 vasopressin receptor, which continues to bind arrestin

while in endosomes, does not recycle back to plasma membrane.

Page 33: Signal Reception: G Protein-Coupled Receptors

Regulation at the G protein levelRegulator of G protein signaling (RGS = GAPs =

GTPase activating proteins) family of proteins (> 20 members) regulate the rate of GTP hydrolysis in the Gα subunit.

Can also attenuate G protein actions that are mediated by βγ subunits, because they can alter the number of βγ available by enhancing the affinity of Gα subunits for the βγ after GTP hydrolysis incr rate of reformation of the heterotimer.

Page 34: Signal Reception: G Protein-Coupled Receptors

Regulation at the G protein level (cont’d)RGS proteins also important in regulating the temporal

characteristics of G protein actions.E.g., RGS proteins accelerate the decay of agonist-

induced activation of GIRK (G protein regulated inward rectifying K channels).

E.g., RGS proteins accelerate desensitization of adrenergic receptor-induced N-type Ca2+ channel currents.

Page 35: Signal Reception: G Protein-Coupled Receptors

D D D D

αα

βα γ

(1) Agonist bindingand G protein

activation

(2) PhosphorylationP P

(3) Arrestinbinding

ArrestinP P

ArrestinP P

Clathrin(4) Clustering inclathrin-coated

pits

(5) EndocytosisEndosomes

ArrestinP P

D

(7) Recycling

(6) Dissociation of agonist:• Dephosphorylation• Sorting between cycling

and lysosomal pathways

(8) Traffic tolysosomes

Lysosomes

Mechanisms of Receptor Regulation

Page 36: Signal Reception: G Protein-Coupled Receptors

Expanding roles for β-arrestins as scaffolds and adaptors in GPCR signaling and trafficking. (Miller & Lefkowitz, 2001. Curr. Opin. Cell Biol. 13:139-145).

Page 37: Signal Reception: G Protein-Coupled Receptors

Another Receptor – G Protein Cycle

Page 38: Signal Reception: G Protein-Coupled Receptors

bg

How G-protein-coupled receptors work (1)

a

extracellular space

cytosol

abg heterotrimeric G-protein

‘7TM’ - receptor

GDP

GDP

N

GTP

Ligand

Page 39: Signal Reception: G Protein-Coupled Receptors

How G-protein-coupled receptors work (2)

inactive

abg

N

GDP

aGTP

P

bg

N

active

Page 40: Signal Reception: G Protein-Coupled Receptors

How G-protein-coupled receptors work (3)

ATP

inactive

inactive

activecAMP

cAMP

Protein kinase A

Phosphorylation of multiple target proteins

bg aGTP

active

Adenylate cyclase

Page 41: Signal Reception: G Protein-Coupled Receptors

Some G-proteins are inhibitoryb-Adrenoceptor

a2-Adrenoceptor

asGTP

ACactive

ACinactive

ai

GTP

Page 42: Signal Reception: G Protein-Coupled Receptors

bg-Subunits of G proteins may have regulatory activity, too

K+

Muscarinic (M2)acetylcholine receptor

Kir

bg

ACinactive

ai

GTP

Page 43: Signal Reception: G Protein-Coupled Receptors

Ga-proteins regulate diverse effector systems

as adenylate cyclase protein kinase A cAMP

ai1 adenylate cyclase protein kinase A cAMP

aq phospholipase C

PIP2 IP3 + DAG protein kinase C

phosphorylation ofmultiple proteins

Ca++

ER

at cGMP phosphodiesterase cGMP

Page 44: Signal Reception: G Protein-Coupled Receptors

Many transmitters have multiple GPCR with different downstream signaling mechanisms

Norepinephrine, a1 IP3 + DAG epinephrine a2 cAMP

b1, b2 cAMP

Dopamine D2 - D4 cAMP D1, D5 cAMP

Acetylcholine M1,, M4, M5 IP3 + DAG M2, M3 cAMP

Page 45: Signal Reception: G Protein-Coupled Receptors

Bivalent muscarinergic agonists

N

NS

NO

OO

N

N S

Nn

Page 46: Signal Reception: G Protein-Coupled Receptors

Dimeric drugs might target heterooligomeric GPCR

Page 47: Signal Reception: G Protein-Coupled Receptors

S

OF3C

CH3

CH3

NH2

PD 81,723 log [PD 81,723]

Cyc

loex

hyla

deon

sine

bi

ndin

g (r

elat

ive)

‘Allosteric’ agonists

Page 48: Signal Reception: G Protein-Coupled Receptors

Cooperative binding to GPCR oligomers may explain the behaviour of pseudo-allosteric

agonists

Page 49: Signal Reception: G Protein-Coupled Receptors

Agonist-specific coupling of a1-adrenergic receptors

Ara

chid

onic

aci

d (%

bas

al)

Inos

itol-P

(% b

asal

)

Log [agonist] (M)

Efficacy: NA = pOCT > mOCT NA > mOCT > pOCT

Page 50: Signal Reception: G Protein-Coupled Receptors

Coupling to multiple G-proteins in the two-state model

GPCRactive

Agonist

GPCRinactive

Antagonist

G-protein A,Effect A

G-protein B,Effect B

Page 51: Signal Reception: G Protein-Coupled Receptors

Agonist-specific coupling implies multiple active states of a GPCR

Agonist B

GPCRinactive

G-protein A,Effect A

G-protein B,Effect B

Agonist AAntagonist