Mechanisms Regulating Endocytosis of Opioid Receptors

描述(申请人提供)：阿片受体是G蛋白偶联受体(GPCRs)超家族的成员，由内源性产生的阿片神经肽和多种成瘾的非肽类药物激活。内源性阿片肽诱导一系列复杂的调节过程，这些过程始于配体激活的受体的快速磷酸化和内吞。非肽类阿片类药物，包括某些重要的阿片类生物碱，如吗啡，已被观察到诱导这些调节过程的程度低于阿片肽激动剂。对完整细胞中野生型受体上发生的特定磷酸化反应知之甚少。此外，特定的磷酸化反应对生理相关的中枢神经系统神经元中受体调节的功能影响还没有明确的定义。拟议的研究使用新的蛋白质生化、质谱学和化学遗传学方法解决了这个问题。其具体目的是1)确定选定的激动剂配体对完整细胞中野生型受体的调节磷酸化的影响；2)确定不同的磷酸化受体种类对受体激动剂选择性调节的功能意义；3)阐明GRK2活性的瞬时升高导致吗啡诱导的UOR内吞持续增加的新机制；以及4)确定HEK293细胞中定义的受体调节机制对吗啡诱导的培养的刺神经元内吞作用的重要性。这些研究特别涉及阿片生物学和成瘾研究领域。它们还可能对理解其他GPCRs的配体依赖调控具有更广泛的意义，这些GPCRs作为一个家族，构成了临床上有用的药物靶点的最大单一组。

DESCRIPTION (provided by applicant): Opioid receptors are members of the large superfamily of G protein-coupled receptors (GPCRs), and are activated both by endogenously produced opioid neuropeptides and by a diverse group of addictive non- peptide drugs. Endogenous opioid peptides induce a complex series of regulatory processes, which begin with rapid phosphorylation and endocytosis of ligand-activated receptors. Non-peptide opioid drugs, including certain important opiate alkaloid such as morphine, have been observed to induce these regulatory processes to a smaller degree than opioid peptide agonists. Little is known about specific phosphorylation reactions occurring on the wild type receptor in intact cells. Furthermore, the functional effects of specific phosphorylation reactions on receptor regulation in physiologically relevant CNS neurons are not defined. The proposed studies address this question using novel protein biochemical, mass spectrometric and chemical genetic approaches. The Specific Aims are to 1) Define the effects of selected agonist ligands on regulatory phosphorylation of wild type receptors in intact cells; 2) Define the functional significance of distinct phosphorylated receptor species to agonist-selective regulation of receptors; 3) Elucidate a novel mechanism by which transient elevation of GRK2 activity produces a sustained increase in morphine- induced uOR endocytosis; and 4) Determine the importance of receptor regulatory mechanisms defined in HEK293 cells to morphine-induced endocytosis in cultured medium spiny neurons. These studies are specifically relevant to the fields of opioid biology and addiction research. They may also have more general implications for understanding ligand-dependent regulation of other GPCRs that, as a family, comprise the largest single group of clinically useful drug targets.