Purification and Mass Spectrometry of Opioid Receptors

描述（由申请人提供）：吸毒成瘾是美国的一个主要医疗问题。阿片类药物成瘾与多种对个人和社会都有害的行为有关。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，但由于慢性阿片类药物使用而导致阿片类药物耐受和依赖的分子机制是复杂的，还有很多需要了解的。阿片类药物的急性管理触发细胞内信号转导，由受体介导的异三聚体G蛋白激活启动。效应器包括腺苷酸环化酶、钾钙离子通道和MAP激酶，所有这些效应器都有助于阿片类药物的药理作用。当在短时间内给予多次剂量时，激动剂的功效迅速下降。这种同源脱敏是由于G蛋白偶联受体激酶（GRKs）对受体的磷酸化导致受体与G蛋白解偶联。阻滞蛋白优先结合GRK磷酸化受体，并阻止G蛋白的进一步活化。长期服用阿片受体激动剂会导致受体下调，包括受体蛋白的蛋白水解和功能性受体数量的减少。很可能是激动剂诱导的阿片受体下调有助于阿片耐受性。PI提供了令人信服的证据，证明泛素/蛋白酶体系统参与基础转换和激动剂诱导的阿片受体下调。脉冲追踪分析显示，激动剂治疗加速了受体的蛋白水解。用蛋白酶体抑制剂（而不是其他蛋白酶抑制剂）预先孵育可阻断激动剂诱导的受体下调。阿片受体的免疫沉淀表明，阿片受体在降解之前是多泛素化的。本研究计划将重点研究阿片受体的纯化和翻译后修饰的质谱分析。阿片受体的磷酸化和泛素化位点将通过光谱分析和位点定向诱变来绘制。用蛋白酶体抑制剂抑制激动剂诱导的下调会减弱耐受性发展的假设将被验证。

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.