A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors.
The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.
Hildebrandt
J D
JD
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Day
R
R
Farnsworth
C L
CL
Feig
L A
LA
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D051379:Mice; D009154:Mutation / Q000235:genetics*; D015689:Oncogene Protein p21(ras) / Q000235:genetics; D012694:Serine / Q000235:genetics; D014407:Tumor Cells, Cultured
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012694:Serine; D001216:Asparagine; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras); D000262:Adenylyl Cyclases; D008274:Magnesium; D007545:Isoproterenol