MECHANISMS UNDERLYING MELATONIN RECEPTOR FUNCTION

药物脱敏在多种疾病的治疗中具有重要的临床意义。脱敏背后的分子事件尚未完全阐明，但可能涉及信号蛋白，特别是受体的修饰。在G蛋白偶联受体（GPRs）超家族中，脱敏似乎涉及G蛋白解偶联对受体的调节。本文将重点研究两种相关褪黑激素受体mt1和mt2的GPRs脱敏的分子基础。mt1褪黑素受体减弱腺苷酸环化酶，主要在大脑中表达。mt2褪黑素受体也减弱腺苷酸环化酶，主要在视网膜中表达。初步数据表明，激动剂暴露导致mt1和mt2褪黑激素受体脱敏。此外，褪黑激素暴露显著改变了转染mt1褪黑激素受体的CHO细胞中的微管蛋白组装。这一建议的假设是，褪黑激素受体/ g蛋白解耦是由于GDP/GTP交换机制受损和/或GTP酶活性增强而导致的，这是脱敏的关键事件。我实验室的长期目标是了解g蛋白偶联受体功能和调节的机制。本提案将通过以下三个具体目标确定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 -移位、GTP酶水解和GTP交换试验来比较和对比转染了mt1 （mt1-CHO）或mt2 （mt2-CHO）褪黑激素受体的CHO细胞中g蛋白的功能。2)我们将使用免疫印迹分析、g -移位、GTP水解和GTP交换试验来比较和对比激动剂暴露对mt1-CHO和mt2-CHO细胞中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水解试验来确定假定的g蛋白功能调节因子，即微管蛋白和RGS4对g蛋白功能的影响。所提出的实验结果将提供g蛋白对GPR脱敏的模型，并将提供一种新的方法来治疗与激动剂暴露后GPR功能丧失相关的各种疾病状态。

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.