The G226A mutant of Gs alpha highlights the requirement for dissociation of G protein subunits.
Adenylylcyclase cannot be activated by hormones or guanine nucleotide analogs in membranes from cells that express the G226A mutant form Gs alpha instead of the wild-type protein. The mutant Gs alpha protein appears incapable of undergoing the conformational change necessary for guanine nucleotide-induced dissociation of the G protein alpha subunit from the beta gamma subunit complex (Miller, R.T., Masters, S.B., Sullivan, K.A., Beiderman, B., and Bourne, H.R. (1988) Nature 334, 712-715). G226A Gs alpha was synthesized in Escherichia coli, purified, and characterized. Examination of the kinetics of dissociation of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) suggests that G226A Gs alpha is incapable of assuming the conformation necessary for high affinity binding of Mg2+ to the alpha subunit-GTP gamma S complex. Associated changes include the failure of Mg2+ and GTP gamma S to confer resistance to tryptic proteolysis upon the protein, to enhance intrinsic tryptophan fluorescence, or to cause dissociation of alpha from beta gamma. However, the GTPase activity of the mutant protein is near normal (at high Mg2+ concentrations), and the protein is capable of activating adenylylcyclase. A similar defect is present in G49V Gs alpha. Failure of G protein subunit dissociation appears to be the explanation for the phenotypic properties of cells that express G226A Gs alpha, and this mutation thus highlights the crucial nature of this reaction as a component of G protein action.
Lee
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Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Taussig
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Gilman
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D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014364:Tryptophan; D004247:DNA; D014357:Trypsin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D008274:Magnesium