Mechanisms Regulating Endocytosis of Opioid Receptors

项目摘要 该提案旨在阐明G蛋白偶联受体（GPCR）如何受到自然调节 产生配体和成瘾性药物，集中在细胞生物学水平上作为连接分子的关键桥梁 系统级事件和理解。GPCR代表了最大的信号受体家族， 包括最大类别的治疗药物靶标，并直接或间接介导 所有成瘾药物的影响。因此，虽然中枢神经系统中的成瘾性药物作用是我们特别关注的， 提出的研究在GPCR家族成员和病理生理学中具有广泛的潜在应用， 流程.我们的首要目标是发展对GPCR调控的基本理解， 基础的细胞生物学，然后阐明其分子基础，并通过这一途径发现新的靶点 以及对成瘾和其他复杂大脑疾病的潜在治疗操作策略， 其特征在于潜在的GPCR信号传导或GPCR依赖性生理调节的紊乱。 上一个资助期的进展集中在确定mu中调节磷酸化的位点上， 阿片受体，在完整的人细胞中表达完整的内源性激酶谱， 本土水平。我们确定了磷酸化的两个关键区域，并进行了详细的细胞生物学研究。 在异源细胞模型和生理学相关的CNS群体中分析其中之一， 衍生神经元在这些研究过程中，我们取得了一些重大的意外进展，包括 构象生物传感器技术的发展，通过异源三聚体G 蛋白质，并发现了一个前所未有的行为arrestin蛋白，这表明一个新的 抑制蛋白的细胞操作模式，在活化GPCR的下游和从活化GPCR解离之后。的 拟议的研究试图发展和扩展这些基本的新观察，并将其发展到 作为新的分子靶点和基于细胞的治疗策略的合理考虑点。

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.