PROTEINS REGULATING ENDOCYTOSIS OF OPIOID RECEPTORS

阿片受体的内吞作用调节信号转导，并被认为参与阿片类药物的作用和成瘾。这一过程在培养的细胞和天然组织中都受到配体的高度调节。 先前已经描述了促进受体内吞作用的磷酸化依赖性机制。初步研究提出了这样的假设，即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。 此外，初步的数据表明，这些机制是相关的阿片受体的调节神经元。 拟议的研究测试这一假设集中在两个不同的机制，这对受体的内吞作用有相反的影响，并在其依赖于受体磷酸化不同。 拟议的实验试图阐明这些机制的生化细节，确定细胞蛋白质介导的，并检查其功能的神经元的后果。 具体目的是（1）表征促进截短、磷酸化缺陷突变型阿片受体快速内吞作用的机制;（2）表征抑制全长阿片受体内吞作用的磷酸化调节机制;（3）鉴定与定义的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，它们可能与了解其他G蛋白偶联受体的内吞调节的替代机制有关。

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.