PROTEINS REGULATING ENDOCYTOSIS OF OPIOID RECEPTORS

阿片受体的内吞作用调节信号转导，被认为与阿片类药物的作用和成瘾有关。无论是在培养细胞中还是在天然组织中，这一过程都受到配体的高度调控。先前已经描述了一种促进受体内吞作用的磷酸化依赖机制。初步研究表明，其他机制有助于在完整细胞中观察到阿片受体内吞作用的生理调节。此外，初步数据表明，这些机制与神经元中阿片受体的调节有关。拟议的研究通过关注两种不同的机制来验证这一假设，这两种机制对受体内吞作用具有相反的影响，并且它们对受体磷酸化的依赖程度不同。拟议的实验试图从生化细节上阐明这些机制，识别介导这些机制的细胞蛋白，并检查它们在神经元中的功能后果。其具体目的是：(1)鉴定一种促进截短的、磷酸化缺陷的突变型阿片受体快速内吞的机制；(2)鉴定一种抑制全长阿片受体内吞作用的磷酸化调节机制；(3)鉴定与特定受体结构域特异相互作用的细胞蛋白，并阐明它们在调节几种细胞类型的阿片受体内吞作用中的作用。这些研究与鸦片类药物作用和成瘾的机制直接相关。此外，它们可能与理解调节其他G蛋白偶联受体的内吞作用的替代机制有关。

Endocytosis of opioid receptors modulates signal transduction and is thought to be involved in opiate drug action and addiction. This process is highly regulated by ligands, both in cultured cells and native tissues. A phosphorylation-dependent mechanism that promotes receptor endocytosis has been described previously. Preliminary studies suggest the hypothesis that additional mechanisms contribute to the physiological regulation of opioid receptor endocytosis observed in intact cells. Furthermore, the preliminary data suggest that these mechanisms are relevant to opiod receptor regulation in neurons. The proposed studies test this hypothesis by focusing on two distinct mechanisms, which have opposite effects on receptor endocytosis and differ in their dependence on receptor phosphorylation. The proposed experiments seek to elucidate these mechanisms in biochemical detail, identify cellular proteins that mediate them, and examine their functional consequences in neurons. The Specific Aims are to (1) characterize a mechanism that promotes rapid endocytosis of a truncated, phosphorylation-deficient mutant opioid receptor; (2) characterize a phosphorylation-regulated mechanism that inhibits endocytosis of full-length opioid receptors; and (3) identify cellular proteins that interact specifically with defined receptor domains and elucidate their role in regulating endocytosis of opioid receptors in several cell types. These studies have direct relevance to mechanisms underlying opiate drug action and addiction. In addition, they may be relevant to understanding alternate mechanisms regulating endocytosis of other G protein-coupled receptors.