Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity.
We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant alpha subunit. The sequence near the carboxyl terminus of Gs alpha was altered to resemble those in Gi alpha polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gs alpha into cyc- (Gs alpha-deficient) S49 cell membranes.
Freissmuth
M
M
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Gilman
A G
AG
D037361:Adenylate Cyclase Toxin; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002772:Cholera Toxin / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013873:Thionucleotides / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D037361:Adenylate Cyclase Toxin; D011994:Recombinant Proteins; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D009243:NAD; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D004247:DNA; D002772:Cholera Toxin; D001120:Arginine; D037342:Pertussis Toxin; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins