Mechanisms Regulating Endocytosis of Opioid Receptors

项目总结 这项建议旨在阐明G蛋白偶联受体(GPCRs)是如何由自然 生产配体和成瘾药物，集中在细胞生物学水平上作为连接分子的关键桥梁 以及系统层面的事件和理解。GPCRs是最大的信号受体家族， 包括最大类别的治疗药物靶点，并直接或间接调解 所有令人上瘾的药物的影响。因此，虽然中枢神经系统中的成瘾药物行动是我们特别关注的，但 所提出的研究具有广泛的潜在应用于GPCR家族成员和病理生理学 流程。我们的总体目标是发展对gpcr监管的基本理解，发现 然后阐明其分子基础，并通过这一途径发现新的靶点 以及对成瘾性和其他复杂脑部疾病的潜在治疗操作的策略 以潜在的gpr信号干扰或gpr依赖的生理调节为特征的。 上一个资助期的进展集中在确定MU的调节磷酸化位点上。 阿片受体，在完整的人类细胞中实现这一点，表达全谱的内源性激酶 原生级别。我们定义了两个关键的磷酸化区域，并进行了详细的细胞生物学研究 在异源细胞模型和中枢神经系统生理相关群体中对其中之一进行了分析。 衍生的神经元。在这些研究过程中，我们取得了一些意想不到的重大进展，包括 构象生物传感器技术的发展，发现了通过异源三聚体G的GPCR信号 来自内体的蛋白质，以及Arrestin蛋白质前所未有的行为的发现，表明了一种新的 阻滞剂的细胞工作模式，在激活的gpr下游和解离后。这个 拟议的研究试图发展和扩展这些基本的新观察，并将它们发展到 理性考虑的观点，作为新的分子靶点和基于细胞的治疗策略。

PROJECT SUMMARY This proposal seeks to elucidate how G protein-coupled receptors (GPCRs) are regulated by naturally produced ligands and addictive drugs, focusing at the cell biological level as the critical bridge linking molecular and systems-level events and understanding. GPCRs represent the largest family of signaling receptors, comprise in aggregate the largest class of therapeutic drug targets, and mediate directly or indirectly the effects of all addictive drugs. Accordingly, while addictive drug action in the CNS is our particular focus, the proposed studies have broad potential application across GPCR family members and pathophysiological processes. Our over-arching goal is to develop a fundamental understanding of GPCR regulation, discovering the underlying cell biology and then elucidating its molecular basis, and through this path discover new targets and strategies for potential therapeutic manipulation of addictive and other complex brain disorders that are characterized by underlying disturbances of GPCR signaling or GPCR-dependent physiological regulation. Progress in the previous funding period focused on defining sites of regulatory phosphorylation in the mu opioid receptor, accomplishing this in intact human cells expressing the full spectrum of endogenous kinases at native levels. We defined two critical regions of phosphorylation and carried out detailed cell biological analysis of one of them, both in a heterologous cell model and a physiologically relevant population of CNS- derived neurons. During the course of these studies we made some major unanticipated progress, including development of conformational biosensor technology, discovery of GPCR signaling via heterotrimeric G proteins from endosomes, and discovery of an unprecedented behavior of arrestin proteins suggesting a new cellular operating mode of arrestins, downstream of and after dissociating from an activating GPCR. The proposed studies seek to develop and extend these fundamental new observations and develop them to the point of rational consideration as new molecular targets and cell-based strategies for therapeutics.