graduate institute of anatomy and cell biology g-protein...

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G-protein-coupled receptors (GPCRs)

黃敏銓[email protected]

Graduate Institute of Anatomy and Cell Biology

台大醫學院解剖學暨細胞生物學研究所

黃敏銓老師實驗室

Graduate Institute of Anatomy and Cell Biology

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The Nobel Prize in Chemistry 2012 is awarded to Brian K. Kobilka (57) and Robert J. Lefkowitz (69) for studies of GPCRs.

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名稱 & 特徵

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CCR5

G protein-coupled receptors (GPCRs) Seven transmembrane receptors (7 TM receptors) Heptahelical receptors Serpentine receptors

名稱

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Single Transmembrane Spanning Heterotrimeric GProtein-Coupled Receptors

Pharmacol Rev. 2004 Sep;56(3):371-85.

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Characteristics of GPCRs

7 transmembrane domains

3 intracellular loops

3 extracellular loopsCytoplasmic

ExtracellularNH2

HOOC

A Transmembrane Intracellular Estrogen Receptor Mediates Rapid Cell Signaling.GPR30 is uniquely localized to the endoplasmic reticulumRevankar CM et al., Science. 2005. 307:1625.

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Genomics: > 800 members

wide range of cellular activities

from yeast to human

50% drug target

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分類

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G-protein-coupled receptor (GPCR) families

• Class A Rhodopsin like

• Class B Secretin like

• Class C Metabotropic glutamate / pheromone

• Class D Fungal pheromone

• Class E cAMP receptors (Dictyostelium)

• Frizzled/Smoothened family

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Family classification of known GPCRs

NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | AUGUST 2002 | 599-608

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | OCTOBER 2002 | 808-820

The three main families of GPCRs

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GRAFS system Five main classes: Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin

Annu Rev Pharmacol Toxicol. 2013;53:127-46

Orphan receptors(unknown ligands)

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訊息傳遞

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http://stke.sciencemag.org/content/vol0/issue2002/images/data/CMP_6634/DC1/science3.jpg, by R. Iyengar's laboratory

http://stke.sciencemag.org/content/vol0/issue2002/images/data/CMP_6634/DC1/science3.jpg�

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Summary of GPCR signalling

NATURE | VOL 415 | 10 JANUARY 2002 206-212

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G protein action

GPCR

ligand1.

GDP βγα

2.

αβγ

GTP

GDP

GPCR(GEF)

second messenger

αβγ

GTP

3.

(GAP)

effectorGPCR

β

4.

αγGDP

Pi

stops

GPCR

G protein cycle for the β2AR–Gs complex

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Nature. 2011 Jul 19;477(7366):549-55.

Kobilka’s group

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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | SEPTEMBER 2002 | 639-650

A classical example of GPCR signalling

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Modified from TRENDS in Pharmacological Sciences Vol.22 No.7 July 2001, 368-376

GPCR G protein effector activities

4 大類

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Regulation of systemic functions by signaling through G protein pathways

31 MAY 2002 VOL 296 SCIENCE, 1636-1639

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | MARCH 2002 | 188

Heterotrimeric G-protein α-subunit subfamilies and their effects

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Desensitization

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Desensitization(receptor level)

(A) Rapid: phosphorylation (sec~min)

(B) Slow: degradation or expression (hours)

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Desensitization(A) Rapid: phosphorylation (sec~min)

1. Second-messenger kinase (PKA, PKC):uncouple GPCR from G proteinligand independentheterologous desensitization

2. GPCR kinase (GRK): GRK1-GRK7binding of β-arrestin and inhibition of G protein interactionligand-activated or activated conformationhomologous desensitization

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DesensitizationGPCR kinase (GRK) phosphorylation


arrestin 1-arrestin 4

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Recycling or degradation?Desensitization: (B) Degradation

SCIENCE VOL 297 26 JULY 2002

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Desensitization

a. GTPase-activating proteinsb. accelerate GTP hydrolysisc. >25 members, for selectivity

RGS (regulator of G-protein signaling)

(Post-receptor level)

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Desensitization by regulator of G-protein signalling (RGS)

NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | MARCH 2002 | 187-197

RGS2 knockout mice:• T-cell activation• Anxiety• Male aggression

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Internalization

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Internalization

1. Desensitization2. Resensitization3. Signal transduction

Functions:

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Internalization

• Clathrin-coated pits–β-arrestin

• Caveolae-mediated– Lipid rafts: signalling

• Uncoated vesicles

Types:

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β-arrestin-dependent internalization of GPCRs

NATURE REVIEWS | NEUROSCIENCE VOLUME 2 | OCTOBER 2001 | 727-733

Function 1: Desensitization

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Function 2: Resensitization

GRP: GPCR specific phosphatase

Pharmacol Rev 53:1–24, 2001

P

P

P

P

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NATURE REVIEWS | NEUROSCIENCE VOLUME 2 | OCTOBER 2001 | 727-733

Function 3: Signal transduction

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TRENDS in Pharmacological Sciences Vol.28 No.8 417

Annu. Rev. Physiol. 2007. 69:511–34

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GPCR signaling via heterotrimeric G proteins from endosomes

J Biol Chem. 2015 Jan 20. [Epub ahead of print]

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G-protein-independent signalling

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G-protein-independent signalling

Regulation of GPCR Signalling by Scaffold Proteins

39Circ Res. 2002;91:672-680

Scaffold Proteins

1

2

3

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1. GPCR/PDZ scaffold interaction

Circ Res. 2002;91:672-680

(calcium channel)

PDZ: PSD-95/Discs-large/ZO-1

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β2 adrenoceptor regulates the Na+-H+ exchanger (NHE)

TRENDS in Biochemical Sciences Vol.26 No.5 May 2001, 291-297

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2. GPCR/non-PDZ scaffold interaction

Circ Res. 2002;91:672-680

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Metabotropic glutamate receptor-mediated Ca2+ release


Ca2+ release

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3. GPCR/β-arrestin interaction

Circ Res. 2002;91:672-680

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β2 Adrenoceptor can mediate the Src kinase pathway


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NATURE | VOL 415 | 10 JANUARY 2002 206-212

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Transactivation

NATURE REVIEWS | CANCER VOLUME 7 | FEBRUARY 2007 | 85

48Trends Endocrinol Metab. 2006 Mar;17(2):48-54

49Trends Endocrinol Metab. 2006 Mar;17(2):48-54

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Oligomerization

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Signal amplification at the receptor level


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Heterodimerization of the GABAB receptor produces a functional receptor


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TRENDS in Pharmacological Sciences Vol.26 No.1 January 2005

Don’t worry ‘B’ happy!:a role for GABABreceptors in anxiety and depressionJohn F. Cryan and KlemensKaupmannNeuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland

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EMBO reports VOL 7 | NO 11 | 2006, p. 1096

Ligand-independent functions of orphan GPCRs in heterodimers

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Bivalent and dimeric ligands


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Drug discoveryGPCR Drugs >$20B annual sales

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GPCR and Diseases

• Obesity• Cardiovascular Disease• Inflammation• Cancer• Diabetes• Alzheimer’s Disaese

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Drug screening

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Key requirements of a ligand screen

• Cell surface expression of GPCRs

• Cellular assays to detect ligand binding

• Libraries of putative natural and synthetic ligands

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Method for “de-orphanizing” GPCRs


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FIRST™ : Functionally Immobilized Receptor Solubilization Technology

Consensus Pharmaceuticals, Inc.

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CARTConstitutively Active Receptor Technology

Arena Pharmaceuticals, Inc.

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DISEASE OVERACTIVE KNOWN GPCRs----------------------------------------------------------------Schizophrenia 5HT2A, D4----------------------------------------------------------------Depression 5HT2A----------------------------------------------------------------Hyperthyroidism Thyrotropin----------------------------------------------------------------Hypertension Angiotensin AT1A----------------------------------------------------------------Asthma Adenosine A1----------------------------------------------------------------Melanoma MC-1----------------------------------------------------------------Retinitis Pigmentosa Rhodopsin----------------------------------------------------------------

Diseases - GPCRs

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Nat Rev Drug Discov. 2011 Jan;10(1):47-60.

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Nat Rev Drug Discov. 2011 Jan;10(1):47-60.

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Representation of ‘inactive’ and ‘active’ GPCR conformations

NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | AUGUST 2002 | 599-608

inactive active

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CARTConstitutively Active Receptor Technology

Inverse agonists: block ligand-independent activity

Selective molecules: one ligand to multiple receptors

Agonists

Allosteric modulators

(Antagonist: block ligand-dependent activity)

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Summary

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Modified from TRENDS in Pharmacological Sciences Vol.22 No.7 July 2001, 368-376

Diversity of G-protein-coupled receptors (GPCRs)

Scaffold proteins

Internalization

signaling

RGS

GRKPKAPKC

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TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001, 396

Treasures throughout the life-cycle of GPCRs

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