Mechanisms Regulating Endocytosis of Opioid Receptors

描述（由申请人提供）：阿片受体，如与其密切相关的肾上腺素能儿茶酚胺受体，在CNS中通过磷酸化和内吞作用进行根本性调节。这些机制是内源性阿片系统的生理稳态的基础，并且可以区分临床相关的非肽类药物如吗啡的作用。我们正在努力了解化学上不同的阿片配体如何产生不同的调节作用。我们的努力是针对分子机制和生理后果。在上一个供资期间开展的工作阐述了一种化学分析方法，该方法基于快速受体纯化和定量质谱法，以解析完整细胞中产生的离散磷酸化受体形式。使用这种方法，我们确定了激动剂选择性肾上腺素能和阿片受体的磷酸化作用。我们还定义了一个特定的磷酸化形式的μ阿片受体，区分吗啡的内吞活性的阿片肽。为了进一步解决机制，我们实施了一个公正的筛选策略，发现新的内吞/再循环调节剂在人类激酶组。为了更精确地研究生理后果，我们合作开发了两种小鼠模型，用于测量和操纵阿片受体磷酸化和内吞作用，在急性制备的脑切片制备。使用这些模型的初步研究表明，GRK 2，一种已知调节阿片受体内吞作用的受体激酶，在培养的细胞模型中，介导阿片类药物脱敏的持续成分，引起慢性吗啡给药在体内的具体要求。拟议的研究旨在：（1）使用分析质谱法解析和定量完整细胞中阿片受体的激动剂选择性磷酸化;（2）通过无偏RNAi筛选鉴定调节阿片受体和肾上腺素能受体的内吞作用的新激酶;（3）确定确定的磷酸化和激酶对HEK 293细胞和培养的神经元中受体内吞作用、表面插入和信号传导的影响;和（4）在完整的脑切片制备物中评估确定的磷酸化和激酶对急性和慢性吗啡调节的功能后果。拟议的研究解决成瘾性药物作用的细胞生物学基础，有助于更普遍地了解部分激动和药物之间的功能选择性的性质，并可能确定新的目标，阿片类药物耐受性或依赖性的药物治疗有用。

DESCRIPTION (provided by applicant): Opioid receptors, like adrenergic catecholamine receptors to which they are closely related, are fundamentally regulated in the CNS by phosphorylation and endocytosis. These mechanisms underlie physiological homeostasis of the endogenous opioid system, and can distinguish the effects of clinically relevant non-peptide drugs such as morphine. We are working to understand how chemically distinct opioid ligands produce different regulatory effects. Our efforts are directed both at molecular mechanism and physiological consequence. Work carried out during the previous funding period elaborated a chemical analytical approach, based on rapid receptor purification and quantitative mass spectrometry, to resolve discrete phosphorylated receptor forms produced in intact cells. Using this approach, we identified agonist-selective effects on phosphorylation of both adrenergic and opioid receptors. We also defined a particular phosphorylated form of the mu opioid receptor that discriminates the endocytic activity of morphine from that of opioid peptide. To further address mechanism, we implemented an unbiased screening strategy for discovering novel endocytic/recycling regulators in the human kinome. To more precisely investigate physiological consequence, we collaboratively developed two mouse models for measuring and manipulating opioid receptor phosphorylation and endocytosis in an acutely prepared brain slice preparation. Preliminary studies using these models suggest a specific requirement for GRK2, a receptor kinase known to modulate opioid receptor endocytosis in cultured cell models, in mediating a sustained component of opioid desensitization that is elicited by chronic morphine administration in vivo. The proposed studies seek to: (1) Resolve and quantify agonist-selective phosphorylation of opioid receptors in intact cells using analytical mass spectrometry; (2) Identify novel kinase(s) that regulate endocytosis of opioid and adrenergic receptors by unbiased RNAi screening; (3) Determine effects of defined phosphorylations and kinases on receptor endocytosis, surface insertion and signaling in HEK293 cells and cultured neurons; and (4) Assess functional consequences of defined phosphorylations and kinases on acute and chronic morphine regulation in an intact brain slice preparation. The proposed studies address the cell biological basis of addictive drug action, contribute more generally to understanding the nature of partial agonism and functional selectivity among drugs, and may identify new targets useful for pharmacotherapy of opiate tolerance or dependence.