g-protein coupled receptors: structure and ...med-fom-apt.sites.olt.ubc.ca/files/2016/04/pcth... ·...
TRANSCRIPT
PCTH 400
G-Protein Coupled Receptors: Structure and FunctionStructure and Function
Dr. Rishi Somvanshi
2405 Wesbrook [email protected]
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 ?