A Mutation in the Putative Mg²⁺-Binding Site of G_s α Prevents Its Activation by Receptors

Abstract

The properties of a G_s α mutant with an Asn substituted for Ser at position 54, designated mutant ⁵⁴Asn α_s, were studied after expression in S49 α_s-deficient (cyc⁻) cells. Ser-54 in α_s is comparable to Ser-17 in Ras, which is involved in binding Mg²⁺ associated with bound nucleotide. ⁵⁴Asn α_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 α_s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTPγS. Guanine nucleotide regulation of mutant α_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 α_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 Ras^H mutant, which has been shown to interfere with endogenous Ras function in cells.