A GTPase-activating protein for the G protein Galphaz. Identification, purification, and mechanism of action.
A GTPase-activating protein (GAP) specific for Galphaz was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. Gz GAP from bovine brain is a membrane protein that is refractory to solubilization with most detergents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of Mr approximately 22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble Galphaz, the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min -1 at 15 degrees C, or to >/=20 min-1 at 30 degrees C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric Gz and m2 muscarinic receptor, Gz GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the Km of the GAP for Galphaz-GTP is 2 nM. Its activity is inhibited by Galphaz-guanosine 5'-O-thiotriphosphate (Galphaz-GTPgammaS) or by Galphaz-GDP/AlF4 with Ki approximately 1.5 nM for both species; Galphaz-GDP does not inhibit. G protein betagamma subunits inhibit Gz GAP activity, apparently by forming a GTP-Galphazbetagamma complex that is a poor GAP substrate. Gz GAP displays little GAP activity toward Galphai1 or Galphao, but its activity with Galphaz is competitively inhibited by both Galphai1 and Galphao at nanomolar concentrations when they are bound to GTPgammaS but not to GDP. Neither phospholipase C-beta1 (a Gq GAP) nor several adenylyl cyclase isoforms display Gz GAP activity.
Wang
J
J
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
Tu
Y
Y
Woodson
J
J
Song
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X
Ross
E M
EM
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D044385:GTP-Binding Protein alpha Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism
D044385:GTP-Binding Protein alpha Subunits; D020690:GTPase-Activating Proteins; D011506:Proteins; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D008274:Magnesium