PROTEINS REGULATING ENDOCYTOSIS OF OPIOID RECEPTORS

阿片受体的内吞作用调节信号转导，被认为与阿片药物作用和成瘾有关。在培养细胞和原生组织中，这一过程受到配体的高度调节。促进受体内吞作用的磷酸化依赖机制先前已被描述过。初步研究提出了在完整细胞中观察到的阿片受体内吞作用的生理调节有其他机制的假设。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。提出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作用，并且对受体磷酸化的依赖性不同。提出的实验旨在阐明这些机制的生化细节，鉴定介导它们的细胞蛋白，并检查它们在神经元中的功能后果。具体目的是：(1)表征一种促进截断的、磷酸化缺陷突变的阿片受体快速内吞的机制；(2)描述了抑制全长阿片受体内吞作用的磷酸化调控机制；(3)鉴定与特定受体结构域特异性相互作用的细胞蛋白，并阐明其在几种细胞类型中调节阿片受体内吞作用的作用。这些研究与阿片类药物作用和成瘾的机制直接相关。此外，它们可能与理解其他G蛋白偶联受体调节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.