Willars G B, McArdle C A, Nahorski S R
Department of Cell Physiology and Pharmacology, University of Leicester, Medical Sciences Building, P.O. Box 138, University Road, Leicester LE1 9HN, U.K.
Biochem J. 1998 Jul 15;333 ( Pt 2)(Pt 2):301-8. doi: 10.1042/bj3330301.
In the present study we have expressed the muscarinic M3 receptor in an immortalized mouse pituitary cell line (alphaT3-1), which expresses an endogenous gonadotropin-releasing hormone (GnRH) receptor, to examine potential differences in acute receptor regulation. Both of these receptors couple to the activation of phosphoinositide-specific phospholipase C (PLC) in these cells and we demonstrate that, despite expression in the same cell background, acute desensitization is a feature of muscarinic M3 receptors but not of GnRH receptors. We show that, when the concentrations of GnRH and methacholine are matched to give approximately equivalent maximal elevations of Ins(1,4,5)P3, the GnRH receptor is able to sustain PLC activity at the initial rate, whereas the muscarinic M3 receptor cannot. Thus PLC-activating G-protein-coupled receptors are able to undergo rapid desensitization in this cell line, indicating that the desensitization profile is receptor-specific rather than cell-specific. This argues strongly that post-receptor regulatory features do not have a prominent role in mediating rapid desensitization in these cells. Furthermore GnRH receptor-mediated PLC activity is sustained despite a marked and persistent depletion in the steady-state level of PtdIns(4,5)P2. In contrast, activation of muscarinic receptors is not sustained despite only a transient decrease in PtdIns(4,5)P2 concentration. Thus, whereas the contribution of PtdIns(4,5)P2 depletion to the temporal profile of receptor-mediated PLC signalling has been difficult to assess, the present results demonstrate that this is unlikely to be of importance in these cells. We suggest that unique structural features of the GnRH receptor result in a lack of appropriate regulatory phospho-acceptor sites and that the absence of agonist-dependent phosphorylation might underlie the lack of acute regulation.
在本研究中，我们在永生化小鼠垂体细胞系（αT3-1）中表达了毒蕈碱型M3受体，该细胞系表达内源性促性腺激素释放激素（GnRH）受体，以研究急性受体调节的潜在差异。这两种受体在这些细胞中均与磷酸肌醇特异性磷脂酶C（PLC）的激活偶联，并且我们证明，尽管在相同的细胞背景中表达，但急性脱敏是毒蕈碱型M3受体的特征，而不是GnRH受体的特征。我们表明，当GnRH和乙酰甲胆碱的浓度匹配以产生大致相等的Ins(1,4,5)P3最大升高时，GnRH受体能够以初始速率维持PLC活性，而毒蕈碱型M3受体则不能。因此，激活PLC的G蛋白偶联受体能够在该细胞系中快速脱敏，这表明脱敏特征是受体特异性的而非细胞特异性的。这有力地表明，受体后调节特征在介导这些细胞中的快速脱敏中没有突出作用。此外，尽管PtdIns(4,5)P2的稳态水平显著且持续耗尽，但GnRH受体介导的PLC活性仍得以维持。相比之下，尽管PtdIns(4,5)P2浓度仅短暂降低，但毒蕈碱受体的激活却不能持续。因此，尽管难以评估PtdIns(4,5)P2耗尽对受体介导的PLC信号传导时间曲线的贡献，但目前的结果表明，这在这些细胞中不太可能具有重要意义。我们认为，GnRH受体独特的结构特征导致缺乏合适的调节磷酸化位点，而缺乏激动剂依赖性磷酸化可能是缺乏急性调节的原因。
