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 ? Receptor coupling to second messenger and Receptor coupling to second messenger and Trafficking 3. Regulation ? Pharmacology and Signaling Dimerization Dimerization 4. 4. Role in Pathological Conditions ? Ro e at o og ca Co d t o s ?
GPCRs (S (Structure and Synthesis) d S h i )
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) • Detects a wide spectrum of extracellular signals , including D t t id t f t ll l i l i l di 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.
Time-line of GPCR Structures Nature 477:549-555 (2011) Nature 494, 185-194 (2013)
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 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 Variation in size is the indication of their diverse structure h d f h d and functions !
G-Protein Coupled Receptors - Classification • Class A (or 1) (Rhodopsin-like) ( ) ( p ) (85% of the GPCR genes) • Class B (or 2) (Secretin receptor family) Cl B ( ) (S i f il ) • 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)
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 ERGIC, ER–Golgi intermediate compartment; COPI: coat protein I, (retrograde transport to the ER); ERAD, ER-associated degradation pathway. Trends in pharmacological Sciences, Volume 29, Issue 10, Pages 528–535
GPCRs Synthesis and Trafficking Large dense-core vesicles (LDCVs) Trends in pharmacological Sciences, Volume 28, Issue 1, 2007, Pages 23–31
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 dimerization coupled-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 ½ YFP ½ YFP • Bioluminescence Resonance Energy Transfer (BRET) DeepBlue DeepBlue Renilla luciferase GFP • Photobleaching FRET (PbFRET) Photobleaching FRET (PbFRET)
Pb-FRET Microscopy Fluorescence Resonance Energy Transfer (FRET) • GFP-tagged receptors • Fluorescently labeled antibodies Fl l l b l d ib di • Fluorescently labeled ligands nsity ty Inten Intensi Time Time
GPCR Functions are Altered upon Dimerization p • GABA • GABA receptors receptors - Receptor Receptor functionality functionality and and sorting sorting (GABA B R1 and GABA B R2) • Dissociation of receptor homodimers is essential for proper receptor trafficking - SSTR2 and d-OR β 2 AR • Inhibition of internalization of the - when heterodimerize with β AR heterodimerize with β 1 AR • Heterodimerization has synergistic y g ( (hSSTR4/hSSTR5) 4/ 5) or result in a non-synergistic effect (hSSTR1/hSSTR5) on cAMP signaling
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 Diseases Cancer Receptor Breast cancer PAR1; EP2; EP4; CXCR4; GPR30 Colon cancer C l EP EP2, EP4; LPA 1; ET receptors; PAR1; Frizzled EP LPA ET t PAR F i l d Head and neck cancer CXCR2; CXCR4; EP receptors; GRPR; PAR1 Small-cell lung cancer g GRPR; NMB-R; CXCR4; CCK 1 ; CCK 2 ; ; 4; 1 ; 2 Non-small-cell lung cancer EP receptors; CXCR2; CXCR4; 1AR; 2AR Ovarian cancer LPA 1 –LPA 3 ; CXCR2 Pancreatic cancer GRPR; CCK 1 ; CCK 2 Parathyroid gland cancer CASR Pituitary cancer tu ta y ca ce TSH receptor; ACTHR S ecepto ; C Prostate cancer PAR1; ET A ; AT1; EP2, EP4; LPA 1 ; B1, B2; GRPR Melanoma MC1R; CXCR2; ET B Basal-cell carcinoma Smoothened Testicular cancer LH receptor Thyroid cancer Thyroid cancer TSH receptor TSH receptor Nature Reviews Cancer 7, 79–94, 2007
GPCRs and Diseases • Nephrogenic diabetes insipides V2 vasopressin receptor • Precocious puberty LH receptor LH receptor • Congenital night blindness Rhodopsin Receptor Rh d i R • Virus entry: y HIV - CCR JCV - 5HT2 R (Serotonin receptor) • Familial gestational hyperthyroidism Thyrotropin receptor
Some Drugs Acting Through GPCRs Biotecnol Apl v.26 n.1 La Habana ene.-mar. 2009
Heterodimers in Pathophysiological Conditions p y g • Acromegaly: Acromegaly: Somatostatin Somatostatin Receptor Receptor 5 5 and and Dopamine 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 expression. i β -Adrenergic β Adrenergic • Cardiac Cardiac Failure: Failure: Angiotensin Angiotensin Receptor Receptor 1/ 1/ Receptor via blocking AT 1 R mediated signaling. • Parkinson’s Disease: Adenosine Receptor 2a and Dopamine Receptor 2 via modulating cell surface expression.
QUESTIONS ?
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