1Growth Hormone & its Receptor (Growth Hormone & its Receptor (extracellular extracellular domains)domains)http://www.cmpharm. u csf.edu/bourne/lab_science/activation.html7.61 Eukaryotic Cell7.61 Eukaryotic CellBiology: Principles andBiology: Principles andPractice, Practice, 20062006Lecture 3Lecture 3G-protein SignalingG-protein SignalingHydrophilic Ligand/Receptor SystemExtracellular Binding Requires Signal‘Transduction’hormonereceptoroutsideinside.hormonereceptorG-ProteinEnzyme oreffector orchannelsecondmessageoutsideinside7-TM or G-Protein Coupled Receptors (GPCR)Majority of transmembrane signaling via hormones,neurotransmitters, sight and smell mediated by GPCRsAbout 2000: ~5% of worm and 3% of mammalian genomes~2000 reported since 1st cloning (1983, bovine opsin). Thehuman genome is now known to encode approximately1000 GPCRs, ~ 400 non-olfactory receptors; ~150 orphanreceptors (~2003); in last 17 years ~15 de-orphanized.23 major families: rhodopsin, calcitonin, and metabotropic.β-adrenergic receptor and rhodopsin 1st purified ancharacterized.Major Drug Targets for Pharmaceutical Industry50% of all modern drugs (~20% of the top 50 best-sellingdrugs) are targeted at GPCRs (including adrenergic,histaminergic, dopaminergic, serotonergic, opiate,cholinergic, etc.) for pain, asthma, inflammation, obesity,cancer, and cardiovascular, metabolic, gastrointestinaland CNS diseases ( e.g., Claritin, Zyprexa, Zantac andCozaar).7-TM or G-Protein Coupled Receptors (GPCR)Focus on G-Protein Coupling(there are others).hormonereceptorG-ProteinEnzyme oreffector orchannelsecondmessageoutsideinsideAdenylate Cyclase SystemHydrophilic hormone/Surface ReceptorATP->cAMPAltered cellular metabolismSome hormones induce, some suppress cyclaseHow do we know what we know?.hormonereceptorG-ProteinEnzyme oreffector orchannelsecondmessageoutsideinsideWe will start from the point at which it was clear that therewere distinct adrenergic receptors (β,α) and glucagonreceptors, and an adenylate cyclase activity that wasactivated by glucagon & β adrenergic receptors and inhibitedby α adrenergic receptors.1971 Nobel Prize to Earl Sutherland for second messagecAMP1994 Nobel Prize: Al Gilman & Martin Rodbell (1971) fordiscovering and working out G-protein mechanism“In World War II [Rodbell] had been in radio communications, typing out Morse code relayed tohim through earphones. Little did he know then the importance that signal transduction wouldplay in his life!” L. Birnbaumer, http://www.sciencemag.org/cgi/content/full/283/5408/16563GTPGDPExchangefactorOccupiedAcceptorEmptyAcceptorGTPGDPiPGTPGeneral Model of G Protein Switchesactivity1. GTP enhanced hormone activation of cyclase andreduction of hormone (glucagon) binding to receptors inmembrane preps. (Rodbell, 1960s) - contaminant of ATP!2. GTP reduces agonist, but not antagonist binding toreceptors (coupling important)3. Nonhydrolyzable GTP analogues (GPPNP) work:(hydrolysis not required)4. Nonhydrolyzable GTP analogues give permanentactivation (a GTPase turns off signal)5. ß-adrenergic receptor-dependent slow GTPase (evidencefor direct role of GTP)6. GTP binding protein purified via detergents by GTPaffinity column away from cyclase, reconstitutes agonist-dependent cyclase in S49 cyc- membranes (receptors andcyclase, but not coupled) and GPPNP-dependent cyclaseactivity (separate subunits) - this is first direct evidence of3 componentsExperimental findings which have lead to the current model:7. Identification of GTP-binding subunit by [32P]GTP-derivedaffinity label and by cholera toxin (which activatessystem)-catalyzed [32P]ADP-ribosylation of GTP-bindingsubunit.8. Purification of receptor and cyclase came after G protein(why?), but finally all reconstituted into liposomes, fullyhormone-sensitive cyclase system requires lipid bilayer.9. Agonist-dependent release of [3H]GDP and binding of[35S]GTPγS (agonist-bound receptor opens up G-bindingsite allowing exchange of GDP and GTP); Kds for variousGTPases are 10-11-10-7 M! very tight binding relative toambient conc. [GTP]>10-4, [GDP]>10-5M; thus, notengineered to respond to GNP levels, not regulated byratio of GTP:GDP10. Kinetics of receptor/G interaction are cyclase independent(R/G & G/C independent).411. Rate constant of activation of Gs-C complex linearlydepends on concentration of the agonist-bound receptor :[k(on-observed) = kon(intrinsic) x [R]total x [H]/(KH+[H]) ; [R]total=total receptor;[H]=agonist concentration; KH=dissociation constant] receptor acts as acatalyst not permanently associated with activatedcomplex: "collision coupling"12. Gs-GDP forms complex with agonist-bound receptor,dissociates when GDP exchanged with GTP or analogue;this dissociation estimated to give 10x amplification ofagonist signal in addition to 100-fold amplification due toslow GTPase activity and high activity of cyclase (similarto light and rhodopsin)13. Receptor-Gx(GDP) complex has higher affinity foragonists than receptor alone. AFFINITY OF RECEPTORFOR ANTAGONIST NOT AFFECTED BY GTP ORANALOGUE; GTP binding reduces affinity of R for agonistand dissociates R-G complex, permits recycling of R,lifetime of G-GTP complex before hydrolysis is manyseconds (GαGTP t1/2~10 sec)Antagonist AgonistKent RS, De Lean A, Lefkowitz RJ. A quantitative analysis of beta-adrenergic receptor interactions: resolution of high and low affinity states of the receptorby computer modeling of ligand binding data. Mol Pharmacol. 1980 Jan;17(1):14-23.Competition of the binding of the β-adrenoceptor antagonistradioligand [3H]dihydroalprenolol to membranes from frogerythrocytes by (a) (–)alprenolol and (b) (–)isoproterenolX= G protein"G" proteins are heterotrimers: α, 39-52 kD, β, 35-36 kD; γ, 8 kDPARTS LIST:ReceptorsEffectors (cyclase)Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. Nature. 2002 Jan10;415(6868):206-12 http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v415/n6868/full/415206a_fs.html5Kristen L. Pierce, Richard T. Premont & Robert J. Lefkowitz Nature Reviews Molecular Cell Biology 3, 639-650 (2002);Signalling SEVEN-TRANSMEMBRANE RECEPTORSGα > 20Gβ > 6Gγ > 12http://mcdb.colorado.edu/courses/3280/lectures/class09-1.htmlFour main G-alpha Familes!"GGDPGs #R sGTPGs #!"GGTP!"GGDPGs #!"GR sGTPGs #GDPSignal Transduction Pathway6GTP!"GGDPGs #!"GR s PGTPGs #GTP!"GGDPGs #!"GR s PGTPGs #CCATPcAMPo f fonSignal Transduction PathwayGTP