MECHANISMS UNDERLYING MELATONIN RECEPTOR FUNCTION

药物脱敏在各种疾病的治疗中具有重要的临床意义。脱敏的分子事件尚未完全阐明，但可能涉及信号蛋白的修饰，特别是受体。在G蛋白偶联受体超家族中，脱敏似乎涉及G蛋白解偶联对受体的调节。这项建议将集中在两个相关的褪黑素受体MT1和MT2的GPRS脱敏的分子基础上。褪黑素的MT1受体减弱腺苷环化酶，主要在大脑中表达。MT2褪黑素受体也能减弱腺苷环化酶，主要在视网膜表达。初步数据表明，激动剂暴露会导致褪黑素受体MT1和MT2的脱敏。此外，褪黑素暴露显著改变了转MT1褪黑素受体的CHO细胞中的微管蛋白组装。支持这一建议的假设是，由于GDP/GTP交换机制受损和/或GTP酶活性增强而导致的褪黑素受体/G蛋白解偶联是脱敏的关键事件。我的实验室的长期目标是了解G蛋白偶联受体功能和调节的潜在机制。这项建议将通过以下三个具体目的来确定G-蛋白在GPR功能和调控中的作用：1)我们将使用免疫印迹分析、G-Shift、GTP酶水解和GTP交换实验来比较和对比转MT1(MT1-CHO)或MT2(MT2-CHO)褪黑素受体的CHO细胞中G-蛋白的功能；2)我们将使用免疫印迹分析、G-Shift、GTP水解和GTP交换实验来比较和对比激动剂暴露对MT1-CHO和MT2-CHO细胞G-蛋白功能的影响。3)我们将通过反义敲除、免疫印迹、GTP交换和GTP水解法来确定可能的G蛋白功能调节剂，即微管蛋白和RGS4对G蛋白功能的影响。这些实验结果将提供一个G蛋白脱敏GPR的模型，并将为治疗与激动剂暴露后GPR功能丧失相关的各种疾病状态提供一种新的方法。

Desensitization to pharmacological agents has important clinical relevance in the treatment of various diseases. The molecular events that underlie desensitization have not been completely elucidated but are likely to involve modification of signaling proteins, particularly receptors. In the superfamily of G- protein coupled receptors (GPRs), desensitization appears to involve regulation of the receptors by G-protein uncoupling. This proposal will focus on the molecular basis of desensitization of GPRs of two related melatonin receptors, mt1 and mt2. The mt1 melatonin receptors attenuate adenylyl cyclase and are primarily expressed in the brain. The mt2 melatonin receptors also attenuate adenylyl cyclase and are primarily expressed in the retina. Preliminary data demonstrate that agonist exposure results in the desensitization of both the mt1 and mt2 melatonin receptors. In addition, melatonin exposure dramatically alters tubulin assembly in CHO cells transfected with the mt1 melatonin receptor. The hypothesis that underlies this proposal is that melatonin receptor/G-protein uncoupling due to impaired GDP/GTP exchange mechanisms and/or to enhanced GTPase activity is a critical event in desensitization. The long term objectives of my laboratory are to understand the mechanisms underlying G-protein coupled receptor function and regulation. This proposal will determine the role that G-proteins play in GPR function and regulation through the following three specific objectives: 1) We will use immunoblot analysis, G-shift, GTPase hydrolysis and GTP exchange assays to compare and contrast the function of G-proteins in CHO cells transfected with either the mt1 (mt1-CHO) or mt2 (mt2-CHO) melatonin receptor, 2) We will use immunoblot analysis, G-shift, GTP hydrolysis and GTP exchange assays to compare and contrast the effects of agonist exposure on G-protein function in mt1-CHO and mt2-CHO cells, and 3) We will determine the effects of putative regulators of G-protein function, that is, tubulin and RGS4, on G-protein function using anti-sense knock down, immunoblot, GTP exchange and GTP hydrolysis assays. The results of the proposed experiments should provide a model of GPR desensitization by G-proteins and will provide a novel approach to treating various disease states associated with a loss of GPR function following agonist exposure.