Mutations of G(sα) designed to alter the reactivity of the protein with bacterial toxins. Substitutions at Arg¹⁸⁷ results in loss of GTPase activity

We have introduced two types of mutations into cDNAs that encode the α subunit of G(s), the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg¹⁸⁷) that is the presumed site of ADP-ribosylation of G(sα) 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 α subunit. The sequence near the carboxyl terminus of G(sα) was altered to resemble those in G(iα) 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 G(sα) into cyc^- (G(sα)-deficient) S49 cell membranes.