G-Protein Coupled Receptors: Structure and Function Structure and - - PowerPoint PPT Presentation

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G-Protein Coupled Receptors: Structure and Function Structure and - - PowerPoint PPT Presentation

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PCTH 400 G-Protein Coupled Receptors: Structure and Function Structure and Function Dr. Rishi Somvanshi 2405 Wesbrook Mall rishiks@mail.ubc.ca 604-827-3672 Learning Objectives 1. GPCR ? Structure and Synthesis 2. Function ?

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SLIDE 1

PCTH 400

G-Protein Coupled Receptors: Structure and Function Structure and Function

  • Dr. Rishi Somvanshi

2405 Wesbrook Mall rishiks@mail.ubc.ca 604-827-3672

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SLIDE 2

Learning Objectives

1. GPCR ?

  • Structure and
  • Synthesis

2. Function ?

  • Receptor coupling to second messenger and

Receptor 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 ?

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SLIDE 3

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

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SLIDE 4

G-Protein Coupled Receptors (GPCRs)

  • Largest and most diverse membrane protein families

Largest and most diverse membrane protein families

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

y g coding genome) D t t id t f t ll l i l i l di

  • Detects a wide spectrum of extracellular signals, including

photons, ions, small organic molecules and entire proteins. Enormous potential for the development of new drugs to target neurological disorders, cancer, cardiac malfunction, asthma, tumours and migraines.

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SLIDE 5

Time-line of GPCR Structures

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

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SLIDE 6

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

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SLIDE 7

Characteristics of all GPCRs Characteristics 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 long

p y 5 3 g h d f h d Variation in size is the indication of their diverse structure and functions !

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SLIDE 8

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

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SLIDE 9

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 transport to the ER); ERAD, ER-associated degradation pathway.

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SLIDE 10

GPCRs Synthesis and Trafficking

Trends in pharmacological Sciences, Volume 28, Issue 1, 2007, Pages 23–31

Large dense-core vesicles (LDCVs)

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SLIDE 11

Sorting of Endocytosed GPCRs

  • Annu. Rev. Pharmacol. Toxicol. 2008.48:537-568.
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SLIDE 12

How GPCRs Function?

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SLIDE 13

Typical cycle of G-Protein Coupled Receptor

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

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SLIDE 14

GPCRs and Signaling Networks

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

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SLIDE 15

cAMP Signaling Pathway

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

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SLIDE 16

Mechanism for the Modulation of Receptor Function DIMERIZATION

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SLIDE 17

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

Nature Reviews Neuroscience 2, 274-286

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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)

DeepBlue Renilla luciferase GFP

Photobleaching FRET (PbFRET)

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SLIDE 19

Pb-FRET Microscopy

Fluorescence Resonance Energy Transfer (FRET)

  • GFP-tagged receptors

Fl l l b l d ib di

  • Fluorescently labeled antibodies
  • Fluorescently labeled ligands

nsity ty Inten Time Intensi Time

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GPCR Functions are Altered upon Dimerization p

  • GABA

receptors Receptor functionality and sorting

  • GABA

receptors

  • Receptor

functionality and sorting (GABABR1 and GABABR2)

  • Dissociation of receptor homodimers is essential for proper

receptor trafficking - SSTR2 and d-OR

  • Inhibition
  • f

internalization

  • f

the β2AR

  • when

heterodimerize with β AR heterodimerize with β1AR

  • Heterodimerization

has synergistic (hSSTR4/hSSTR5)

  • r

y g ( 4/ 5) result in a non-synergistic effect (hSSTR1/hSSTR5) on cAMP signaling

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Role of Dimerization in the Transport of GPCRs

Nature Reviews Neuroscience 2, 274-286

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SLIDE 22

Taste Qualities and the Taste Receptors

J Cell Biol 2010;190:285-296

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SLIDE 23

Role in Pathological Conditions

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GPCRs and Diseases

Cancer Receptor Breast cancer PAR1; EP2; EP4; CXCR4; GPR30 C l EP EP LPA ET t PAR F i l d Colon 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; CCK2 g ; ; 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; ACTHR tu 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

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GPCRs and Diseases

  • Nephrogenic diabetes insipides

V2 vasopressin receptor

  • Precocious puberty

LH receptor LH receptor

  • Congenital night blindness

Rh d i R Rhodopsin Receptor

  • Virus entry:

y HIV - CCR JCV - 5HT2 R (Serotonin receptor)

  • Familial gestational hyperthyroidism

Thyrotropin receptor

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Some Drugs Acting Through GPCRs

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

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Heterodimers in Pathophysiological Conditions p y g

  • Acromegaly:

Somatostatin Receptor 5 and Dopamine Acromegaly: Somatostatin Receptor 5 and Dopamine receptor 2 agonist (Dopastatins) in regulation of Tumors.

  • AIDS:

Chemokine receptor 2 (CCR2) / CCR5 or C-X-C chemokine receptor type 4 (CXCR4) via modulating CXCR4 i expression.

  • Cardiac

Failure: Angiotensin Receptor 1/ β-Adrenergic Cardiac Failure: Angiotensin Receptor 1/ β Adrenergic Receptor via blocking AT1R mediated signaling.

  • Parkinson’s Disease: Adenosine Receptor 2a and Dopamine

Receptor 2 via modulating cell surface expression.

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SLIDE 28

QUESTIONS ?