Identification of both G_i2 and a novel, immunologically distinct, form of G_o in rat myometrial membranes

Abstract

Immunoblotting of rat myometrial membranes with an antiserum (SGl) which recognizes the a-subunits of both G,l and G,2 indicated the presence of detectable levels of an apparently single form of some 40 kDa. A second antiserum (LE2) specific for G,2 also recognized this protein, confirming its identity. Immunoblotting of the myometrial membranes with a series of antipeptide (OCl, IMl, ONl) and polyclonal (RV3) antisera, all of which recognize rat brain G,, produced a more complex pattern. Antisera OCl and ON1 recognized a single polypeptide which essentially comigrated with rat brain G,. In contrast, antisera RV3 and IMI did not recognize the myometrial protein. These data provide evidence for the presence of Gi2 and of a novel G-protein, related immunologically to G,, in rat myometrial membranes.Guanine nucleotide-binding protein; Pertussis toxin; (Myometrium, Brain)

1,4‐Dihydropyridines modulate GTP hydrolysis by Go in neuronal membranes

“…Both the C-and N-terminal regions of Go~ may be important in this modulation as the stimulation of GTPase by ago:~.ist dihydropyridines was completely eliminated by pretreatment of membranes with specific antisera raised against either the C-or N-terminal regions of Gocz. Previously, both antisera have demon- strated a high specificity and selectivity to recognize a 39 kDa protein corresponding to Go~ while not crossreacting with G~ [19]. Our results have been extended by showing that Bay K 8644 stimulates GTPase in membranes prepared from cultured cerebellar granule neurons, and that this effect is prevented by pretreatment of the cells with pertussis toxin (M. Silver, M.1.S.…”

“…Stimulation of GTPase by the dihydropyridines was not affected by AS7 which recognizes Gi [19], strongly suggesting that L-type Ca 2+ channels do not interact directly with Gi. Baclofen-induced stimulation of GTPase was reduced substantially by anti-G~0~ antisera, but also was reduced partially by anti-Go0~ antisera, suggesting that GABAa receptors interact with Gi, and to a lesser extent with Go, to stimulate a high affinity GTPase.…”

“…The specific antisera used were OCI or OC2 and ONI, raised against C-and N-terminal peptides, respectively, of Go¢~, and AS7, which is directed against a C-terminal peptide sequence from G~ [19]. The stimulatory effect of dihydropyridine agonists on GTPas¢ was completely eliminated by pretreatment of membranes with the antiGoCZ antisera.…”

Different forms of Go alpha mRNA arise by alternative splicing of transcripts from a single gene on human chromosome 16.

“…In the case of the a subunits, four species of G.. arise by alternative splicing of transcripts from a single gene (10,29,31,53), whereas the several members of the Gi family, designated Gij,l, Gia,2, and Giot3, are products of separate genes (9,13,16,26,27,29). The question of whether there is one or multiple forms of G., (gene symbol GNA01) has been raised in several contexts (7,20,25,38,48,49,63). At least three individual species of G.0, mRNA transcripts can be identified on Northern (RNA) analysis using GOO-specific probes (8,40,41).…”

“…seen for mRNA coding for G, subunits. Evidence of multiple forms of G., protein has been reported (7,20,30,38). Using Mono Q chromatography and two-dimensional electrophoresis, Goldsmith et al (20) separated two proteins of 39 and 39.5 kDa from bovine brain that reacted similarly with four different G oa-specific antisera.…”