Expression and analysis of Gs alpha mutants with decreased ability to activate adenylylcyclase.
We have constructed mutants of the alpha subunit of Gs in an attempt to identify sites in the protein that are important for its interaction with adenylylcyclase. Some residues specific for those G proteins that activate adenylylcyclase were replaced with residues characteristic of Gi alpha. Mutant proteins were expressed in Escherichia coli, and two of these were purified to homogeneity and characterized in detail. Mutation of trp263, leu268, or arg269 caused a significant loss of the capacity of Gs alpha to stimulate adenylylcyclase, and the triple mutant had less than 1% of the ability of wild type Gs alpha to activate the enzyme. Guanine nucleotide binding and GTP hydrolysis by the mutant proteins were unaltered, as was guanosine 5'-3-O-(thio)triphosphate-induced enhancement of intrinsic tryptophan fluorescence. Mutant proteins also appeared to have a reduced affinity for the G protein beta gamma subunit complex. Secondary structure analysis and comparison with the structure of p21ras suggests that the region of Gs alpha that we have identified is part of a loop that may be involved in interaction of the protein with adenylylcyclase. Although these residues are essential for activation of adenylylcyclase, they are not sufficient to do this when placed in the context of another G protein alpha subunit.
Itoh
H
H
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Gilman
A G
AG
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D004247:DNA; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases