Activating mutations in the NH2‐ and COOH‐terminal moieties of the Gsα subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation

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Abstract
The α subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The αs and αi2 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 αs polypeptide (Osawa et al: Cell 63:697–706, 1990). The NH2-terminal half of the αs polypeptide encodes domains regulating βγ interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2-or COOH-terminal coding sequence of αs substituted with the corresponding αi2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the αs polypeptide. Mutation of either the amino- or carboxy-terminus results in an αs polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated αs polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTPγS activation of adenylyl cyclase. In membrane preparations from cells expressing the various αs mutants, COOH-terminal mutants, but not NH2-terminal αs mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTPγS and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the αs polypeptides. Thus, mutation at NH2-and COOH-termini influence the rate of αs activation, but only the COOH-terminus appears to be involved in the regulation of the αs polypeptide activation domain that interacts with adenylyl cyclase.