Activating mutations in the NH 2 ‐ and COOH‐terminal moieties of the G s α subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation

The α subunit polypeptides of the G proteins G s and G i2 stimulate and inhibit adenylyl cyclase, respectively. The α s and α i2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH‐terminal moiety of the α s polypeptide (Osawa et al: Cell 63:697–706, 1990). The NH 2 ‐terminal half of the α s polypeptide encodes domains regulating βγ interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH 2 ‐or COOH‐terminal coding sequence of α s substituted with the corresponding α i2 sequence were used to introduce multi‐residue non‐conserved mutations in different domains of the α s polypeptide. Mutation of either the amino‐ or carboxy‐terminus results in an α s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated α s polypeptides having mutations in either the NH 2 ‐ or COOH‐terminus demonstrate an enhanced rate of GTPγS activation of adenylyl cyclase. In membrane preparations from cells expressing the various α s mutants, COOH‐terminal mutants, but not NH 2 ‐terminal α s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTPγS and fluoride ion. Neither mutation at the NH 2 ‐ nor COOH‐terminus had an effect on the GTPase activity of the α s polypeptides. Thus, mutation at NH 2 ‐and COOH‐termini influence the rate of α s activation, but only the COOH‐terminus appears to be involved in the regulation of the α s polypeptide activation domain that interacts with adenylyl cyclase.