Gs couples thyrotropin-releasing hormone receptors expressed in Xenopus oocytes to phospholipase C

Abstract

Coupling of thyrotropin-releasing hormone (TRH) receptors to individual G-proteins has been studied in Xenopus oocytes injected with receptor cRNA and antisense oligonucleotides to mRNA encoding different G-protein α- and β-subunits. Injection of antisenses which target mRNA sequences shared by several G-protein α or βγ polypeptides effectively blocked Ca2+-dependent Cl- currents induced by TRH through activation of phospholipase C. Three different αs-specific antisense oligonucleotides complementary to sequences located in different positions along the coding region of the as protein mRNA were highly effective in inhibiting TRH-induced responses. Anti-αo, -αq, -αi, or -αz oligonucleotides were not able to modify the TRH-evoked response. In contrast, anti-αo, but not anti-αs, oligonucleotides blocked the response to serotonin in oocytes injected with serotonin 5-HT1c receptor cRNA. Cholera toxin catalyzed the [32P]ADP-ribosylation of 40-42- and 50-52-kDa proteins in GH3 cell plasma membranes. [32P]ADP-ribosylation of oocyte membranes with the toxin labeled several proteins. These include a single 50-55-kDa substrate, which is clearly diminished in membranes from anti-αs-injected oocytes. Amplification of oocyte RNA in a polymerase chain reaction system and sequencing of the amplified products demonstrated that anti-αs oligonucleotides selectively recognize the message for the Xenopus αs polypeptide. It is concluded that Gs, but not Go, Gq, Gi, or Gz, 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.