Cellular Pharmacology of Kappa Opioid Receptor

描述(申请人提供)：激活(Kappa)阿片受体可产生多种作用，包括止痛、烦躁和利水，kappa激动剂可用作止痛剂、利水剂和止痒药物。Kappa阿片受体通过百日咳毒素敏感的G蛋白偶联，影响多种效应器。重复或持续服用Kappa激动剂会导致耐受和依赖。部分耐受性可以在受体水平上解释。申请者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。经选择性激动剂U50,488H作用后，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。是否有信号指导内化受体的分类还不是很清楚。这一应用的中心假设是，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、运输和信号传递中发挥重要作用。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。与EBP50/NHERF-1相互作用的HKOR中的残基将通过突变研究来确定。将评估受体磷酸化和泛素化对其与EBP50/NHERF-1结合的影响，并研究EBP50/NHERF在U50,488H诱导的Na+，H+交换刺激中的作用。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GABARAPL 1的区域和参与相互作用的HKOR。将从hkor的转运、表面表达、配体结合和信号转导等方面研究hkor-GABARAPL1相互作用的功能意义。(3)界定泛素化在香港港元的信号和贩运中所扮演的角色。我们将研究不同激动剂和受体磷酸化对HKOR泛素化的影响。泛素化的位置将通过诱变来确定。泛素化对HKOR的功能特性、调节和贩运的影响将被调查。阐明EBP50/NHERF-1和GABARAPL 1及其受体泛素化在HKOR转运和信号传递中的功能意义将有助于更好地了解HKOR的细胞生物学，并将对开发kappa激动剂和拮抗剂作为治疗药物具有重要意义。此外，这种理解将对其他G蛋白偶联受体具有重要的影响，因为这些分子可能对其他受体发挥类似的作用。

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.