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

Abstract

The alpha subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The alpha s and alpha 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 alpha s polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH2-terminal half of the alpha s polypeptide encodes domains regulating beta gamma interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2- or COOH-terminal coding sequence of alpha s substituted with the corresponding alpha i2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the alpha s polypeptide. Mutation of either the amino- or carboxy-terminus results in an alpha s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated alpha s polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTP gamma S activation of adenylyl cyclase. In membrane preparations from cells expressing the various alpha s mutants, COOH-terminal mutants, but not NH2-terminal alpha s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTP gamma S and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the alpha s polypeptides. Thus, mutation at NH2- and COOH-termini influence the rate of alpha s activation, but only the COOH-terminus appears to be involved in the regulation of the alpha s polypeptide activation domain that interacts with adenylyl cyclase.

Differentiation of F9 Teratocarcinoma Stem Cells to Primitive Endoderm Is Regulated by the Giα2/Gsα Axis via Phospholipase C and Not Adenylylcyclase

“…3). Confirming earlier studies on adenylylcyclase in other cells (15,16), expression of G s␣ and G s␣ G225T significantly enhanced cyclic AMP accumulation. Substitution of G s␣ sequence with G i␣2 (1-7) , G i␣2 (1-64) , G i␣2 , and G i␣2 sequences attenuated the enhanced response observed with expression of either G s␣ or G s␣ G225T.…”

“…The patterns for cyclic AMP accumulation by clones expressing the various subunits and chimeras were similar for the basal, isoproterenol-stimulated (100 M), and forskolin-stimulated (50 M) conditions.These data demonstrate that, when stably expressed in the context of F9 teratocarcinoma cells, G s␣ stimulates, G i␣2 inhibits, and substitution of G s␣ with increasing N-terminal regions of G i␣2 attenuates activation of adenylylcyclase. Thus, the G-proteins expressed have retained the ability to recognize receptor, bind and exchange GTP, and to activate one known effector, adenylylcyclase, as previously noted (15,16).…”

“…1) were inserted in the pCW1 expression vector driven by the SV40 early promoter and harboring the neomycin resistance gene. N-terminal substitutions of G s␣ by analogous sequences of G i␣2 were employed (15), as was the substitution of the C-terminal 38 residues of G s␣ with the corresponding C-terminal 36 residues of G i␣2 (16). Similarly, wild-type and the constitutively active G225T mutant of G s␣ and wild-type and the constitutively active Q205L mutant of G i␣2 were expressed also.…”

Existence of Multiple Novel Gsalpha Splice Variants in Acute Leukemia Patients

“…Certain mutations of Gsa subunit, which alter the GTPase cycle, have been shown to be involved in such tumors as pituitary and thyroid tumors, with mutations often occurring at the same 2 critical sites as mentioned above: amino acid residues 201 and 227. These mutations induce a stabilization of Gsa in the active conformation by inhibiting its intrinsic GTPase activity and lead to a constitutive activation of adenylyl cyclase, thereby promoting cell proliferation (20,21). Based on this mechanism, the aberrant splicings with in-frame deletions presented in our study also raise the possibility of potentially oncogenic Gsa subunits de cient in GTPase activity.…”

Identification of Amino Acid Residues of Gsα Critical to Repression of Adipogenesis

“…2B). Earlier studies have demonstrated that Gs␣-Gi␣2 chimera with these types of substitutions retain their capacity to bind GTP and transduce signaling (17,24).…”