de la Peña P, del Camino D, Pardo L A, Domínguez P, Barros F
Departamento de Biología Functional, Facultad de Medicina, Universidad de Qviedo, Spain.
J Biol Chem. 1995 Feb 24;270(8):3554-9. doi: 10.1074/jbc.270.8.3554.
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.
已在注射了受体cRNA和针对编码不同G蛋白α和β亚基的mRNA的反义寡核苷酸的非洲爪蟾卵母细胞中，研究了促甲状腺激素释放激素（TRH）受体与单个G蛋白的偶联情况。注射针对几种G蛋白α或βγ多肽共有的mRNA序列的反义寡核苷酸，可有效阻断TRH通过激活磷脂酶C诱导的Ca(2+)依赖性Cl-电流。三种不同的αs特异性反义寡核苷酸，与αs蛋白mRNA编码区不同位置的序列互补，在抑制TRH诱导的反应方面非常有效。抗αo、-αq、-αi或-αz寡核苷酸无法改变TRH诱发的反应。相比之下，抗αo寡核苷酸（而非抗αs寡核苷酸）可阻断注射了5-羟色胺5-HT1c受体cRNA的卵母细胞对5-羟色胺的反应。霍乱毒素催化了GH3细胞质膜中40 - 42 kDa和50 - 52 kDa蛋白质的[32P]ADP核糖基化。用该毒素对卵母细胞膜进行[32P]ADP核糖基化标记了几种蛋白质。其中包括一个单一的50 - 55 kDa底物，在注射了抗αs的卵母细胞的膜中明显减少。在聚合酶链反应系统中对卵母细胞RNA进行扩增并对扩增产物进行测序表明，抗αs寡核苷酸可选择性识别非洲爪蟾αs多肽的信使RNA。结论是，在卵母细胞中表达的TRH受体与磷脂酶C的激活以及随后肌醇1,4,5-三磷酸依赖性刺激Ca(2+)依赖性Cl-电流的偶联是通过Gs，而非Go、Gq、Gi或Gz实现的。
