Gs couples thyrotropin-releasing hormone receptors expressed in Xenopus oocytes to phospholipase C.
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 alpha- and beta-subunits. Injection of antisenses which target mRNA sequences shared by several G-protein alpha or beta gamma polypeptides effectively blocked Ca(2+)-dependent Cl- currents induced by TRH through activation of phospholipase C. Three different alpha s-specific antisense oligonucleotides complementary to sequences located in different positions along the coding region of the alpha s protein mRNA were 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 anti-alpha s, 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-alpha s-injected oocytes. Amplification of oocyte RNA in a polymerase chain reaction system and sequencing of the amplified products demonstrated that anti-alpha s oligonucleotides selectively recognize the message for the Xenopus alpha s 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 Ca(2+)-dependent Cl- currents.
de la Peña
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Departamento de Biología Functional, Facultad de Medicina, Universidad de Qviedo, Spain.
del Camino
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Pardo
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Domínguez
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Barros
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D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D009865:Oocytes; D018075:RNA, Complementary; D011985:Receptors, Serotonin / Q000235:genetics; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*; D014981:Xenopus
D002712:Chlorides; D016376:Oligonucleotides, Antisense; D018075:RNA, Complementary; D011985:Receptors, Serotonin; D018025:Receptors, Thyrotropin-Releasing Hormone; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium