G(S) COUPLES THYROTROPIN-RELEASING-HORMONE RECEPTORS EXPRESSED IN XENOPUS OOCYTES TO PHOSPHOLIPASE-C

Coupling of thyrotropin-releasing hormone (TRH) receptors to individua l G-proteins has been studied in Xenopus oocytes injected with recepto r cRNA and antisense oligonucleotides to mRNA encoding different G-pro tein alpha- and beta-subunits. Injection of antisenses which target mR NA sequences shared by several G-protein alpha or beta gamma polypepti des effectively blocked Ca2+-dependent Cl- currents induced by TRH thr ough activation of phospholipase C. Three different alpha(s)-specific antisense oligonucleotides complementary to sequences located in diffe rent positions along the coding region of the alpha(s) protein mRNA we re highly effective in inhibiting TRH-induced responses. Anti-alpha(o) , -alpha(q), -alpha(i), or -alpha(z) oligonucleotides were not able to modify the TRH-evoked response. In contrast, anti-alpha(o), but not a nti-alpha(s), oligonucleotides blocked the response to serotonin in oo cytes injected with serotonin 5-HT1(c) receptor cRNA. Cholera toxin ca talyzed the [P-32]ADP-ribosylation of 40-42- and 50-52-kDa proteins in GH(3) cell plasma membranes, [P-32]ADP-ribosylation of oocyte membran es with the toxin labeled several proteins. These include a single 50- 55-kDa substrate, which is clearly diminished in membranes from anti-a lpha(s)-injected oocytes. Amplification of oocyte RNA in a polymerase chain reaction system and sequencing of the amplified products demonst rated that anti-alpha(s) oligonucleotides selectively recognize the me ssage for the Xenopus alpha(s) polypeptide. It is concluded that G(s), but not G(o), G(q), G(i), or G(z), couples TRH receptors expressed in oocytes to activation of phospholipase C and subsequent inositol 1,4, 5-trisphosphate-dependent stimulation of Ca2+-dependent Cl- currents.