Growth Hormone its Receptor extracellular extracellular domains 7 61 Eukaryotic Cell Biology Principles and Practice 2005 Lecture 3 G protein Signaling http www cmpharm ucsf edu bourne lab scienc e activation html Hydrophilic Ligand Receptor System Extracellular Binding Requires Signal Transduction hormone receptor outside inside hormone receptor outside 7 TM or G Protein Coupled Receptors GPCR inside G Protein Enzyme or effector or second channel Majority of transmembrane signaling via hormones message neurotransmitters sight and smell mediated by GPCRs About 2000 5 of worm and 3 of mammalian genomes 2000 reported since 1st cloning 1983 bovine opsin The human genome is now known to encode approximately 1000 GPCRs 400 non olfactory receptors 150 orphan receptors 2003 in last 17 years 15 de orphanized 1 7 TM or G Protein Coupled Receptors GPCR 3 major families rhodopsin calcitonin and metabotropic adrenergic receptor and rhodopsin 1st purified an characterized Major Drug Targets for Pharmaceutical Industry 50 of all modern drugs 20 of the top 50 best selling drugs are targeted at GPCRs including adrenergic histaminergic dopaminergic serotonergic opiate cholinergic etc for pain asthma inflammation obesity cancer and cardiovascular metabolic gastrointestinal and CNS diseases e g Claritin Zyprexa Zantac and Cozaar Focus on G Protein Coupling there are others hormone receptor outside inside G Protein second message Enzyme or effector or channel Adenylate Cyclase System Hydrophilic hormone Surface Receptor ATP cAMP Altered cellular metabolism Some hormones induce some suppress cyclase hormone How do we know what we know receptor outside inside G Protein second message Enzyme or effector or channel We will start from the point at which it was clear that there were distinct adrenergic receptors and glucagon receptors and an adenylate cyclase activity that was activated by glucagon adrenergic receptors and inhibited by adrenergic receptors 1971 Nobel Prize to Earl Sutherland for second message cAMP 1994 Nobel Prize Al Gilman Martin Rodbell 1971 for discovering and working out G protein mechanism In World War II Rodbell had been in radio communications typing out Morse code relayed to him through earphones Little did he know then the importance that signal transduction would play in his life L Birnbaumer http www sciencemag org cgi content full 283 5408 1656 2 General Model of G Protein Switches GTP activity Exchange factor GTP GTP GDP Occupied Acceptor GDP iP Empty Acceptor Experimental findings which have lead to the current model 1 GTP enhanced hormone activation of cyclase and reduction of hormone glucagon binding to receptors in membrane preps Rodbell 1960s contaminant of ATP 2 GTP reduces agonist but not antagonist binding to receptors coupling important 3 Nonhydrolyzable GTP analogues GPPNP work hydrolysis not required 4 Nonhydrolyzable GTP analogues give permanent activation a GTPase turns off signal 5 adrenergic receptor dependent slow GTPase evidence for direct role of GTP 6 GTP binding protein purified via detergents by GTP affinity column away from cyclase reconstitutes agonistdependent cyclase in S49 cyc membranes receptors and cyclase but not coupled and GPPNP dependent cyclase activity separate subunits this is first direct evidence of 3 components 7 Identification of GTP binding subunit by 32P GTP derived affinity label and by cholera toxin which activates system catalyzed 32P ADP ribosylation of GTP binding subunit 8 Purification of receptor and cyclase came after G protein why but finally all reconstituted into liposomes fully hormone sensitive cyclase system requires lipid bilayer 9 Agonist dependent release of 3H GDP and binding of 35 S GTP S agonist bound receptor opens up G binding site allowing exchange of GDP and GTP Kds for various GTPases are 10 11 10 7 M very tight binding relative to ambient conc GTP 10 4 GDP 10 5M thus not engineered to respond to GNP levels not regulated by ratio of GTP GDP 10 Kinetics of receptor G interaction are cyclase independent R G G C independent 3 11 Rate constant of activation of Gs C complex linearly depends on concentration of the agonist bound receptor k on observed kon intrinsic x R total x H K H H R total total receptor H agonist concentration KH dissociation constant receptor acts as a catalyst not permanently associated with activated complex 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 of agonist signal in addition to 100 fold amplification due to slow GTPase activity and high activity of cyclase similar to light and rhodopsin 13 Receptor Gx GDP complex has higher affinity for agonists than receptor alone AFFINITY OF RECEPTOR FOR ANTAGONIST NOT AFFECTED BY GTP OR ANALOGUE GTP binding reduces affinity of R for agonist and dissociates R G complex permits recycling of R lifetime of G GTP complex before hydrolysis is many seconds G GTP t1 2 10 sec Competition of the binding of the adrenoceptor antagonist radioligand 3H dihydroalprenolol to membranes from frog erythrocytes by a alprenolol and b isoproterenol Antagonist Agonist Kent RS De Lean A Lefkowitz RJ A quantitative analysis of beta adrenergic receptor interactions resolution of high and low affinity states of the receptor by computer modeling of ligand binding data Mol Pharmacol 1980 Jan 17 1 14 23 PARTS LIST Receptors G proteins are heterotrimers 39 52 kD 35 36 kD 8 kD Effectors cyclase Rockman HA Koch WJ Lefkowitz RJ Seven transmembrane spanning receptors and heart function Nature 2002 Jan 10 415 6868 206 12 http www nature com cgi taf DynaPage taf file nature journal v415 n6868 full 415206a fs html 4 G 20 G 6 G 12 Kristen L Pierce Richard T Premont Robert J Lefkowitz Nature Reviews Molecular Cell Biology 3 639 650 2002 Signalling SEVEN TRANSMEMBRANE RECEPTORS Four main G alpha Familes http mcdb colorado edu courses 3280 lectures class09 1 html Rs G GsGDP Signal Transduction Pathway G Rs GTP Gs G GsGDP G GTP GTP Gs GDP 5 Rs G GsGDP Signal Transduction Pathway G GTP GTP Gs P Rs G GsGDP G off GTP C GTP Gs P C on cAMP ATP Signal Transduction Pathway Rs G GsGDP Ri G off GTP P GTP Gi GTP Gs Gi C G GTP C ATP GDP G P on cAMP G is GTP binding protein and GTPase GTP hydrolysis used as a timing device For AR H R t1 2 1 sec G GTP t1 2 10 sec G Protein s Features kcatGTPase for G 2 4 min slow but much faster than ras kcatGTPase ras 0 02 min almost off