MECHANISMS UNDERLYING MELATONIN RECEPTOR FUNCTION

褪黑激素受体功能的机制

• 批准号: 2729010
• 负责人: PAULA ANN WITT-ENDERBY
• 金额: $ 9.26万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-20 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2729010
• 关键词: CHO cells; G protein; antisense nucleic acid; guanine nucleotide exchange factors; guanosinetriphosphatases; hormone receptor; melatonin; protein isoforms; protein structure function; receptor coupling; receptor sensitivity; tubulin; western blottings

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.

对药物的脱敏在各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（尤其是受体）的修饰。 在 G 蛋白偶联受体 (GPR) 超家族中，脱敏似乎涉及通过 G 蛋白解偶联来调节受体。该提案将重点关注两种相关褪黑激素受体 mt1 和 mt2 GPR 脱敏的分子基础。 mt1 褪黑激素受体可减弱腺苷酸环化酶，主要在大脑中表达。 mt2 褪黑激素受体也会减弱腺苷酸环化酶，并且主要在视网膜中表达。 初步数据表明，激动剂暴露会导致 mt1 和 mt2 褪黑激素受体脱敏。 此外，褪黑激素暴露显着改变了转染 mt1 褪黑激素受体的 CHO 细胞中微管蛋白的组装。 该提议的假设是，由于 GDP/GTP 交换机制受损和/或 GTP 酶活性增强，褪黑激素受体/G 蛋白解偶联是脱敏的关键事件。 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。该提案将通过以下三个具体目标确定 G 蛋白在 GPR 功能和调节中发挥的作用： 1) 我们将使用免疫印迹分析、G-shift、GTPase 水解和 GTP 交换测定来比较和对比转染 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-shift、GTP 水解和 GTP 交换测定，以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，以及 3) 我们将使用反义敲低、免疫印迹、GTP 交换确定 G 蛋白功能的假定调节剂（即微管蛋白和 RGS4）对 G 蛋白功能的影响 和 GTP 水解测定。 所提出的实验结果应提供 G 蛋白 GPR 脱敏模型，并将提供一种新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。

项目成果

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence.

• DOI: 10.1111/j.1600-079x.2008.00644.x
• 发表时间: 2009-03
• 期刊: Journal of pineal research
• 影响因子: 10.3
• 作者: Jarzynka MJ;Passey DK;Johnson DA;Konduru NV;Fitz NF;Radio NM;Rasenick M;Benloucif S;Melan MA;Witt-Enderby PA
• 通讯作者: Witt-Enderby PA

Knock-down of RGS4 and beta tubulin in CHO cells expressing the human MT1 melatonin receptor prevents melatonin-induced receptor desensitization.

在表达人 MT1 褪黑激素受体的 CHO 细胞中敲低 RGS4 和 β 微管蛋白可防止褪黑激素诱导的受体脱敏。

• DOI: 10.1016/j.lfs.2004.08.002
• 发表时间: 2004
• 期刊: Life sciences.
• 作者: Witt-Enderby,PA;Jarzynka,MJ;Krawitt,BJ;Melan,MA
• 通讯作者: Melan,MA