PCTH 400

G-Protein Coupled Receptors: Structure and FunctionStructure and Function

Dr. Rishi Somvanshi

2405 Wesbrook Mallrishiks@mail.ubc.ca

604-827-3672

Learning Objectives

1. GPCR ? Structure and Synthesis

2. Function ? Receptor coupling to second messenger andReceptor coupling to second messenger and Trafficking

3. Regulation ? Pharmacology and Signaling Dimerization Dimerization

4. Role in Pathological Conditions ?4. Ro e at o og ca Co d t o s ?

GPCRs (S d S h i )(Structure and Synthesis)

G-Protein Coupled Receptors (GPCRs)

•Largest and most diverse membrane protein familiesLargest and most diverse membrane protein families

•Encoded by more than 800 genes (or ≈4% of the entire protein-y gcoding genome)

D t t id t f t ll l i l i l di•Detects a wide spectrum of extracellular signals, includingphotons, ions, small organic molecules and entire proteins.

Enormous potential for the development of new drugs to targetneurological disorders, cancer, cardiac malfunction, asthma,tumours and migraines.

Time-line of GPCR Structures

Nature 494, 185-194 (2013)Nature 477:549-555 (2011)

Characteristics of GPCRs

•N terminal segment•N-terminal segment

•Seven Transmembrane Domains which constitute

i. TM Core

ii. Three exoloops

iii. Three Cytoloops

•C-terminal segment

Pharmacol Ther. 2004 Jul;103(1):21-80

Characteristics of all GPCRsCharacteristics of all GPCRs

•N-terminal segments has 7-595 aa

•C-terminal segments contains 12-359 aa

•Each of the 7 TMs is generally composed of 20 27 aa•Each of the 7 TMs is generally composed of 20-27 aa

•Loops are normally 5-230 aa longp y 5 3 g

h d f h dVariation in size is the indication of their diverse structure and functions !

G-Protein Coupled Receptors - Classification

•Class A (or 1) (Rhodopsin-like) ( ) ( p )

(85% of the GPCR genes)

Cl B ( ) (S i f il )•Class B (or 2) (Secretin receptor family)

•Class C (or 3) (Metabotropic glutamate/pheromone)

•Class D (or 4) (Fungal mating pheromone receptors)

•Class E (or 5) (Cyclic AMP receptors)•Class E (or 5) (Cyclic AMP receptors)

•Class F (or 6) (Frizzled/Smoothened)

GRAFS (Glutamate Rhodopsin Adhesion Frizzled/Taste2GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, Secretin)

GPCRs Synthesis and Trafficking

COPII, coat protein II, transport of proteins from the rough ER to the Golgi apparatus;ERGIC ER Golgi intermediate compartment; COPI: coat protein I (retrograde transport

Trends in pharmacological Sciences, Volume 29, Issue 10, Pages 528–535

ERGIC, ER–Golgi intermediate compartment; COPI: coat protein I, (retrograde transportto the ER); ERAD, ER-associated degradation pathway.

GPCRs Synthesis and Trafficking

Trends in pharmacological Sciences, Volume 28, Issue 1, 2007, Pages 23–31Large dense-core vesicles (LDCVs)

Sorting of Endocytosed GPCRs

Annu. Rev. Pharmacol. Toxicol. 2008.48:537-568.

How GPCRs Function?

Typical cycle of G-Protein Coupled Receptor

Nature Volume: 477, Pages:549–555, 2011

GPCRs and Signaling Networks

Trends in pharmacological Sciences, Volume 22, Issue 7, 1 July 2001, Pages 368–376

cAMP Signaling Pathway

O'Connor, C. M. & Adams, J. U. Essentials of Cell Biology. Cambridge, MA: NPG Education, 2010.

Mechanism for the Modulation of Receptor Function

DIMERIZATION

Molecular determinants of G-protein-coupled receptor dimerizationcoupled-receptor dimerization

Nature Reviews Neuroscience 2, 274-286

Biophysical Techniques to Study GPCR Dimerization

• Colocalization

• Co-immunoprecipitation /Western blot analysis

• Bimolecular fluorescence complementation (BiFC)p

• Bioluminescence Resonance Energy Transfer (BRET)

½ YFP ½ YFP

DeepBlue

• Photobleaching FRET (PbFRET)

DeepBlueRenilla luciferase GFP

Photobleaching FRET (PbFRET)

Pb-FRET MicroscopyFluorescence Resonance Energy Transfer (FRET)• GFP-tagged receptors

Fl l l b l d ib di• Fluorescently labeled antibodies• Fluorescently labeled ligands

nsityty

Inte

n

Time

Intensi

Time

GPCR Functions are Altered upon Dimerizationp

•GABA receptors Receptor functionality and sorting•GABA receptors - Receptor functionality and sorting(GABABR1 and GABABR2)

•Dissociation of receptor homodimers is essential for properreceptor trafficking - SSTR2 and d-OR

•Inhibition of internalization of the β2AR - whenheterodimerize with β ARheterodimerize with β1AR

•Heterodimerization has synergistic (hSSTR4/hSSTR5) ory g ( 4/ 5)result in a non-synergistic effect (hSSTR1/hSSTR5) on cAMPsignaling

Role of Dimerization in the Transport of GPCRs

Nature Reviews Neuroscience 2, 274-286

Taste Qualities and the Taste Receptors

J Cell Biol 2010;190:285-296

Role in Pathological Conditions

GPCRs and DiseasesCancer Receptor

Breast cancer PAR1; EP2; EP4; CXCR4; GPR30

C l EP EP LPA ET t PAR F i l dColon cancer EP2, EP4; LPA1; ET receptors; PAR1; Frizzled

Head and neck cancer CXCR2; CXCR4; EP receptors; GRPR; PAR1

Small-cell lung cancer GRPR; NMB-R; CXCR4; CCK1; CCK2g ; ; 4; 1; 2

Non-small-cell lung cancer EP receptors; CXCR2; CXCR4; 1AR; 2AR

Ovarian cancer LPA1–LPA3 ; CXCR2

Pancreatic cancer GRPR; CCK1; CCK2

Parathyroid gland cancer CASR

Pituitary cancer TSH receptor; ACTHRtu ta y ca ce S ecepto ; C

Prostate cancer PAR1; ETA; AT1; EP2, EP4; LPA1; B1, B2; GRPR

Melanoma MC1R; CXCR2; ETB

Basal-cell carcinoma Smoothened

Testicular cancer LH receptor

Thyroid cancer TSH receptor

Nature Reviews Cancer 7, 79–94, 2007

Thyroid cancer TSH receptor

GPCRs and Diseases• Nephrogenic diabetes insipides

V2 vasopressin receptor

• Precocious pubertyLH receptorLH receptor

• Congenital night blindnessRh d i RRhodopsin Receptor

• Virus entry: yHIV - CCR JCV - 5HT2 R (Serotonin receptor)

• Familial gestational hyperthyroidismThyrotropin receptor

Some Drugs Acting Through GPCRs

Biotecnol Apl v.26 n.1 La Habana ene.-mar. 2009

Heterodimers in Pathophysiological Conditionsp y g

• Acromegaly: Somatostatin Receptor 5 and DopamineAcromegaly: Somatostatin Receptor 5 and Dopaminereceptor 2 agonist (Dopastatins) in regulation of Tumors.

• AIDS: Chemokine receptor 2 (CCR2) / CCR5 or C-X-Cchemokine receptor type 4 (CXCR4) via modulating CXCR4

iexpression.

• Cardiac Failure: Angiotensin Receptor 1/ β-AdrenergicCardiac Failure: Angiotensin Receptor 1/ β AdrenergicReceptor via blocking AT1R mediated signaling.

• Parkinson’s Disease: Adenosine Receptor 2a and DopamineReceptor 2 via modulating cell surface expression.

QUESTIONS ?