Cellular Pharmacology of Kappa Opioid Receptor

Kappa 阿片受体的细胞药理学

• 批准号: 7064864
• 负责人: LEE-YUAN LIU-CHEN
• 金额: $ 33.07万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7064864
• 关键词: binding sites; biological signal transduction; cell line; genetic manipulation; guanine nucleotide binding protein; maleimides; microtubule associated protein; opioid receptor; pharmacology; phosphorylation; posttranslational modifications; protein localization; protein protein interaction; protein structure function; protein transport; receptor binding; receptor expression; sodium hydrogen exchanger; stimulant /agonist; transfection; transport proteins; tubulin; ubiquitin

DESCRIPTION (provided by applicant): Activation of (kappa) opioid receptors produces many effects, including analgesia, dysphoria and water diuresis and kappa agonists may be useful as analgesics, water diuretics and antipruritic drugs. Kappa opioid receptors are coupled through pertussis toxin-sensitive G proteins to affect a variety of effectors. Repeated or continuous administration of Kappa agonists leads to tolerance and dependence. Part of tolerance can be accounted for at the receptor level. The applicant's long term objectives are to understand at the molecular level the biochemical events that occur following chronic exposure to opioid drugs. After exposure to the selective agonist U50, 488H, the human kappa opioid receptor (hkor) undergoes internalization and the internalized receptors are recycled to plasma membranes or sorted to lysosomes and proteasomes for degradation. Whether there are signals directing the sorting of the internalized receptors is not well understood. The central hypothesis of this application is that the hkor is associated with EBP50/NHERF-1 and GABARAPL1 and undergoes ubiquitination, all of which play important roles in the regulation, trafficking and signaling of the receptor. The specific aims are as follows. (1) To investigate the interaction of the hkor with EBP50/NHERF-1, a PDZ domain-containing protein. The residues in the hkor involved in the interaction with EBP50/NHERF-1 will be determined by mutagenesis studies. Effects of receptor phosphorylation and ubiquitination on its association with EBP50/NHERF-1 will be assessed and the role of EBP50/NHERF in the U50,488H-induced stimulation of Na+,H+-exchange will be examined. (2) To examine the interaction of the hkor with GABARAPL 1. Whether there are interactions between GABARAPL1 and tubulin, N-ethylmaleimide-sensitive factor, EBP50/NHERF-1, rab5 or rab7 will be investigated. The regions of GABARAPL 1 and the hkor involved in the interaction will be determined. The functional significance of the hkor-GABARAPL1 interaction will be studied in terms of trafficking, surface expression, ligand binding and signal transduction of the hkor. (3) To delineate the role of ubiquitination in the signaling and trafficking of the hkor. Effects of different agonists and receptor phosphorylation on ubiquitination of the hkor will be examined. The sites of ubiquitination in the hkor will be determined by mutagenesis. The impact of ubiquitination on the functional properties, regulation and trafficking of the hkor will be investigated. Elucidation of the functional significance of EBP50/NHERF-1 and GABARAPL 1 and receptor ubiquitination in the trafficking and signaling of the hkor will provide better understanding of cell biology of the hkor and will have implications for development of kappa agonists and antagonists as therapeutic agents. In addition, such understanding will have important ramifications for other G protein coupled receptors since these molecules may play similar roles for other receptors.

描述（由申请人提供）：（κ）阿片受体的激活产生许多作用，包括镇痛、烦躁和利水，并且κ激动剂可用作镇痛药、利尿剂和止痒药。 Kappa 阿片受体通过百日咳毒素敏感 G 蛋白偶联，影响多种效应器。重复或连续施用 Kappa 激动剂会导致耐受性和依赖性。部分耐受性可以在受体水平上解释。申请人的长期目标是在分子水平上了解长期接触阿片类药物后发生的生化事件。暴露于选择性激动剂 U50、488H 后，人 kappa 阿片受体 (hkor) 发生内化，内化的受体被回收到质膜或分选到溶酶体和蛋白酶体进行降解。是否存在指导内化受体分类的信号尚不清楚。该申请的中心假设是hkor与EBP50/NHERF-1和GABARAPL1相关并经历泛素化，所有这些都在受体的调节、运输和信号传导中发挥重要作用。具体目标如下。 (1) 研究hkor与含有PDZ结构域的蛋白EBP50/NHERF-1的相互作用。 hkor中参与与EBP50/NHERF-1相互作用的残基将通过诱变研究来确定。将评估受体磷酸化和泛素化对其与 EBP50/NHERF-1 关联的影响，并检查 EBP50/NHERF 在 U50,488H 诱导的 Na+、H+ 交换刺激中的作用。 (2)考察hkor与GABARAPL 1的相互作用。考察GABARAPL1与微管蛋白、N-乙基马来酰亚胺敏感因子、EBP50/NHERF-1、rab5或rab7之间是否存在相互作用。将确定参与相互作用的 GABARAPL 1 和 hkor 的区域。我们将从 hkor 的运输、表面表达、配体结合和信号转导方面研究 hkor-GABARAPL1 相互作用的功能意义。 (3) 描述泛素化在hkor信号传导和运输中的作用。将检查不同激动剂和受体磷酸化对 hkor 泛素化的影响。 hkor 中的泛素化位点将通过诱变来确定。将研究泛素化对 hkor 的功能特性、监管和贩运的影响。阐明 EBP50/NHERF-1 和 GABARAPL 1 以及受体泛素化在 hkor 运输和信号传导中的功能意义将有助于更好地了解 hkor 的细胞生物学，并将对作为治疗剂的 κ 激动剂和拮抗剂的开发产生影响。此外，这种理解将对其他 G 蛋白偶联受体产生重要影响，因为这些分子可能对其他受体发挥类似的作用。