Purification and Mass Spectrometry of Opioid Receptors

描述（由申请人提供）：药物成瘾是美国的一个主要医疗问题。阿片类药物成瘾与各种对个人和社会有害的行为有关。虽然在过去的三十年里，人们对阿片类药物的药理学和信号转导有了很多了解，但由于长期使用阿片类药物而导致阿片类药物耐受性和依赖性的分子机制是复杂的，还有很多东西有待了解。阿片类药物的急性给药触发细胞内信号转导，由受体介导的异源三聚体G蛋白激活启动。效应子包括腺苷酸环化酶、钾和钙离子通道以及MAP激酶，所有这些都有助于阿片类药物的药理学作用。当在短时间内给予多次剂量时，激动剂效力迅速降低。这种同源脱敏是由于受体与G蛋白解偶联，由于G蛋白偶联受体激酶（GRKs）的受体磷酸化。Arrestin优先结合GRK磷酸化受体，并阻止G蛋白的进一步激活。阿片激动剂的长期给药导致受体下调，涉及受体蛋白的蛋白水解和功能性受体数量的伴随减少。激动剂诱导的阿片受体下调极有可能有助于阿片耐受。PI提供了令人信服的证据表明，泛素/蛋白酶体系统参与阿片受体的基础转换和激动剂诱导的下调。脉冲追踪分析表明，激动剂治疗加速受体的蛋白水解。与蛋白酶体抑制剂预孵育，而不是其他蛋白酶抑制剂，阻断激动剂诱导的受体下调。阿片受体的免疫沉淀显示阿片受体在降解之前是多泛素化的。本研究计划将集中于阿片受体的纯化和使用质谱分析的翻译后修饰。阿片受体的磷酸化和泛素化位点将通过质谱分析和定点突变进行定位。将检验用蛋白酶体抑制剂抑制激动剂诱导的下调将减弱耐受性发展的假设。

DESCRIPTION (provided by applicant): Drug addiction is a major medical problem in the United States. Opioid addiction is associated with a variety of behaviors that are detrimental to both the individual and society. While much has been learned about opioid pharmacology and signal transduction over the last three decades, the molecular mechanisms that are responsible for opioid tolerance and dependence due to chronic opioid drug use are complex and much remains to be learned. Acute administration of opioids triggers intracellular signal transduction, initiated by receptor-mediated heterotrimeric G protein activation. Effectors include adenylyl cyclase, potassium and calcium ion channels, and MAP kinase, all of which contribute to the pharmacological effects of opioids. Agonist efficacy diminishes rapidly when multiple doses are given over a short period of time. This homologous desensitization is due to uncoupling of the receptor from the G protein, due to receptor phosphorylation by G protein-coupled receptor kinases (GRKs). Arrestin binds preferentially to GRK phosphorylated receptors and precludes further activation of G proteins. Chronic administration of opioid agonists results in receptor down regulation, involving proteolysis of the receptor protein and a concomitant decrease in the number of functional receptors. It is highly probable that agonist-induced down regulation of opioid receptors contributes to opioid tolerance. The PI has provided compelling evidence that the ubiquitin/proteasome system is involved in basal turnover and agonist-induced down regulation of opioid receptors. Pulse-chase analysis revealed that agonist treatment accelerates proteolysis of the receptor. Preincubation with proteasome inhibitors, but not other protease inhibitors, blocked agonist-induced receptor down regulation. Immunoprecipitation of opioid receptors revealed that opioid receptors are polyubiquitinated prior to degradation. This research proposal will focus on the purification of opioid receptors and analysis of post-translational modification using mass spectrometry. Sites of phosphorylation and ubiquitination of opioid receptors will be mapped by mas spectrometric analysis and site-directed mutagenesis. The hypothesis that inhibition of agonist-induced down regulation with proteasome inhibitors will attenuate the developments of tolerance will be tested.