RECEPTOR-STIMULATED GTP?S BINDING BY G PROTEINS

By JAMES PEREZ

CONTENTS STRUCTURAL BASIS OF EFFECTOR REGULATION AND SIGNAL TERMINATION IN HETEROTRIMERIC G PROTEINS I INTRODUCTION AND SCOPE II A SELECTIVE SURVEY OF GA PROTEIN STRUCTURE AND FUNCTION III MECHANISMS OF EFFECTOR RECOGNITION AND REGULATION BY GAGTP IV SIGNAL TERMINATION: THE MECHANISM OF GTP HYDROLYSIS AND CONFORMATIONAL DEACTIVATION V SIGNAL TERMINATION THROUGH GAPS AND EFFECTOR GAP DOMAINS VI CONCLUSIONS REFERENCES HOW DO RECEPTORS ACTIVATE G PROTEINS? I INTRODUCTION II TOWARD A MODEL OF THE RECEPTOR–G PROTEIN COMPLEX III MOLECULAR BASIS FOR G PROTEIN ACTIVATION IV SUMMARY AND CONCLUSIONS SOME MECHANISTIC INSIGHTS INTO GPCR ACTIVATION FROM DETERGENT-SOLUBILIZED TERNARY COMPLEXES ON BEADS I PERSPECTIVES II SURVEY OF EXPERIMENTAL APPROACHES AND REPRESENTATIVE DATA III ANALYSIS OF SOLUBLE RECEPTOR TERNARY COMPLEX ASSEMBLIES IV GUANINE NUCLEOTIDE ACTIVATION OF TERNARY COMPLEX: SOME DYNAMIC ASPECTS OF STRUCTURE AND REACTIVITY ACTIVATION OF G PROTEIN–COUPLED RECEPTORS I INTRODUCTION II STRUCTURAL AND MECHANISTIC HOMOLOGY AMONG GPCRS III CONFORMATIONAL STATES IV ACTIVATION BY AGONISTS V CONCLUDING REMARKS KINETIC ANALYSIS OF G PROTEIN–COUPLED RECEPTOR SIGNALING USING FLUORESCENCE RESONANCE ENERGY TRANSFER IN LIVING CELLS I INTRODUCTION II ASSAYS AND METHODS III RESULTS AND DISCUSSION IV CONCLUSIONS REGULATION OF RHO GUANINE NUCLEOTIDE EXCHANGE FACTORS BY G PROTEINS I INTRODUCTION II RGS-RHOGEFS III MECHANISMS OF REGULATION IV PHYSIOLOGICAL FUNCTION OF THE RGS-RHOGEFS STRUCTURAL BASIS OF EFFECTOR REGULATION AND SIGNAL TERMINATION IN HETEROTRIMERIC GA PROTEINS I INTRODUCTION AND SCOPE A A COMMON GA:EFFECTOR INTERFACE B FUNCTIONAL CONSEQUENCES OF GA:EFFECTOR BINDING A STRUCTURE OF THE GROUND STATE FOR GTP HYDROLYSIS B REACTION TRAJECTORY FOR GTP HYDROLYSIS A DEACTIVATION OF GAQ BY PLCB B RGS GAPS C SYNERGY BETWEEN RGS AND EFFECTOR DOMAINS D DEACTIVATION OF GA13 BY THE AGAP ELEMENT OF P115RHOGEF ABBREVIATIONS APCPP ADENOSINE 50?(AB?METHYLENE)?TRIPHOSPHATE ATPAS ADENOSINE 50?(A?THIO)?TRIPHOSPHATE (RP) CGMP CYCLIC GUANOSINE 30,50?MONOPHOSPHATE CHD CYCLASE HOMOLOGY DOMAIN DEP DISHEVELED/EGL?10/PLEXTRIN HOMOLOGY DOMAIN DH DBL HOMOLOGY FTIR FOURIER TRANSFORM INFRARED RAMAN GA G PROTEIN A SUBUNIT GAP GTPASE?ACTIVATING PROTEIN GDI GUANINE NUCLEOTIDE DISSOCIATION INHIBITOR GEF GUANINE NUCLEOTIDE EXCHANGE FACTOR GPCR G PROTEIN–COUPLED RECEPTOR GPPNP GUANOSINE 50?(BG?IMINO)?TRIPHOSPHATE GRK G PROTEIN RECEPTOR KINASE GTPGS GUANOSINE 50?(G?THIO)?TRIPHOSPHATE KIE KINETIC ISOTOPE EFFECT C2 DOMAIN OF TYPE II MAC ABSTRACT THIS CHAPTER ADDRESSES, FROM A MOLECULAR STRUCTURAL PERSPECTIVE GAINED FROM EXAMINATION OF X?RAY CRYSTALLOGRAPHIC AND BIOCHEMICAL DATA, THE MECHANISMS BY WHICH GTP?BOUND GA SUBUNITS OF HETEROTRIMERIC G PROTEINS RECOGNIZE AND REGULATE EFFECTORS THE MECHANISM OF GTP HYDROLYSIS BY GA AND RATE ACCELERATION BY GAPS ARE ALSO CONSIDERED THE EFFECTOR RECOGNITION SITE IN ALL GA HOMOLOGUES IS FORMED ALMOST ENTIRELY OF THE RESIDUES EXTENDING FROM THE C?TERMINAL HALF OF A2 (SWITCH II) TOGETHER WITH THE A3 HELIX AND ITS JUNCTION WITH THE B5 STRAND EFFECTOR BINDING DOES NOT INDUCE SUBSTANTIAL CHANGES IN THE STRUCTURE OF GAGTP EFFECTORS ARE STRUCTURALLY DIVERSE DIFFER- ENT EFFECTORS MAY RECOGNIZE DISTINCT SUBSETS OF EFFECTOR?BINDING RESIDUES OF THE SAME GA PROTEIN SPECI?CITY MAY ALSO BE CONFERRED BY DIFFERENCES IN THE MAIN CHAIN CONFORMATION OF EFFECTOR?BINDING REGIONS OF GA SUBUNITS SEVERAL GA REGULATORY MECHANISMS ARE OPERATIVE IN THE REGULATION OF GMP PHOSPO- DIESTERASE, GAT SEQUESTERS AN INHIBITORY SUBUNIT GAS IS AN ALLOSTERIC ACTIVATOR AND INHIBITOR OF ADENYLYL CYCLASE, AND GAI IS AN ALLOSTERIC INHIBITOR GAQ DOES NOT APPEAR TO REGULATE GRK, BUT IS RATHER SEQUESTERED BY IT GTP HYDROLYSIS TERMINATES THE SIGNALING STATE OF GA THE BINDING ENERGY OF GTP THAT IS USED TO STABILIZE THE GA:EFFECTOR COMPLEX IS DISSIPATED IN THIS REACTION CHEMICAL STEPS OF GTP HYDROLYSIS, SPECI?CALLY, FORMATION OF A DISSOCIATIVE TRANSITION STATE, IS RATE LIMITING IN RAS, A MODEL G PROTEIN GTPASE, EVEN IN THE PRESENCE OF A GAP; HOWEVER, THE ENERGY OF ENZYME REORGANIZATION TO PRODUCE A CATALYTICALLY ACTIVE CONFORMATION APPEARS TO BE SUBSTANTIAL IT IS POSSIBLE THAT THE COLLAPSE OF THE SWITCH REGIONS, ASSOCIATED WITH GA DEACTIVATION, ALSO RECEPTOR STIMULATED GTPγS BINDING BY G PROTEINS