活细胞条件下G蛋白偶联受体激酶在孤啡肽受体脱敏和内化中的作用

Opioid receptor desensitization is considered to be one molecular mechanism of opioids tolerance and addiction, in which G protein-coupled receptor kinases (GRKs)-catalyzed receptor phosphorylation plays a key role. Orphanin FQ receptor (ORL1),a newly discovered opioid system, mediates some physiological activities which are different with classical opioid systems's. Orphanin FQ can block the analgesic effects of classical opioid peptides. ORL1 and classic opioid receptors can form heterodimer, which can affect the desensitization and resensitization of the classic opioid. Therefore, orphanin FQ and its receptor may provide new ideas for the study of opioid tolerance, addiction and the treatment of many diseases. Bioluminescence resonance energy transfer (BRET) technology is a newly method to explore protein-protein interaction. We have been successfully applied BRET to study the dimerization of κ opioid receptors and Apelin receptors. In this project, we aim to use BRET combining with co-immunoprecipitation, confocal microscopy, gene mutation, immunoblotting and other technology to analyze the role of GRKs subtypes in ORL1 phosphorylation, desensitization and internalization process and to determine the phosphorylation sites of ORL1. This research will provide helpful data to clarify the molecular mechanism of the interaction of GRKs and ORL1 in living cells which can make ORL1 as a potential new target for drug development.

阿片受体脱敏被认为是阿片类药物产生耐受性和成瘾性分子机制之一，而G蛋白偶联受体激酶(GRKs)催化的受体磷酸化在其中起关键作用。孤啡肽及其受体(ORL1)是新近发现的阿片系统，其介导的某些生理活动有别于经典阿片系统，可阻断经典阿片肽的镇痛作用，激活的ORL1可与经典阿片受体形成异二聚体而影响其脱敏和复敏，为研究阿片类药物的耐受、成瘾及多种疾病的治疗提供了新思路。生物发光共振能量转移(BRET)技术是目前研究蛋白与蛋白相互作用的新技术，本课题组前期已成功应用BRET研究κ阿片受体和Apelin受体间相互作用。在此基础上，本项目拟应用BRET结合免疫共沉淀、共聚焦显微镜、基因突变、免疫印记等技术系统地分析GRKs各亚型在ORL1磷酸化、脱敏、内化过程中的作用，确定ORL1被GRKs磷酸化的位点，阐明活细胞状态下二者相互作用的分子机理，为确定孤啡肽受体成为潜在的新的药物作用靶点提供依据。

阿片受体脱敏被认为是阿片类药物产生耐受性和成瘾性的机制之一，而G蛋白偶联受体激酶(GRKs)催化的受体磷酸化在其中起关键作用。生物发光共振能量转移（bioluminescence resonance energy transfer, BRET）技术是近十年来出现的一种新的检测蛋白质-蛋白质相互作用的技术，可在活细胞状态下实时、动态检测激动剂诱导的GPCRs与细胞内GRKs之间的相互作用，并且无损伤性，因此能够进行相互作用动力学的研究。本课题应用BRET结合免疫共沉淀、共聚焦显微镜、基因突变、免疫印记等技术系统地分析GRKs各亚型在ORLⅠ磷酸化、脱敏、内化过程中的作用，确定了ORLⅠ被GRKs磷酸化的位点，阐明了活细胞状态下二者相互作用的分子机理，这将有助于使孤啡肽受体成为新的药物靶点。