Molecular Mechanisms of mu-Opioid Receptor Processing

mu-阿片受体加工的分子机制

• 批准号: 6793955
• 负责人: TERRI L GILBERT
• 金额: $ 2.82万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-02-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6793955
• 关键词: G protein coupled receptor kinase; SDS polyacrylamide gel electrophoresis; arrestins; autoradiography; casein kinase; cell line; confocal scanning microscopy; drug tolerance; flow cytometry; fluorescence resonance energy transfer; opioid receptor; phosphorylation; polymerase chain reaction; postdoctoral investigator; protein kinase; receptor expression; site directed mutagenesis; stimulant /agonist; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Opioid receptors are the major site of action of pain relieving medications such as morphine and its derivatives. These drugs are potent analgesics, although they can produce addiction and tolerance making their clinical use problematic. The rationale of this study is to understand the mechanisms behind opioid receptor desensitization and internalization with the intent to begin to unravel the molecular mechanisms behind tolerance. Desensitization and internalization of the receptor require the interactions of protein kinases and arrestins. The aims of this project are to determine the agonist-dependent specificity of kinase and arrestin action in both the desensitization and internalization of the mu-opioid receptor. This study will utilize wild-type and mutants of the mu-opioid receptor that have putative phosphorylation sites deleted. These receptors will be labeled with fluorescent proteins and transfected into tissue culture cell-lines. Antibodies specific for the phosphorylated forms of the receptor will be developed and used in Western analysis and immunocytochemistry. Fluorescence Resonance Energy Transfer (FRET) will be used to determine the temporal interactions of the receptor and accessory proteins and will be quantified using both flow cytometry and confocal imaging.

描述（由申请人提供）： 阿片受体是吗啡及其衍生物等止痛药物的主要作用部位。这些药物是有效的镇痛药，尽管它们会产生成瘾和耐受性，从而导致其临床使用存在问题。这项研究的基本原理是了解阿片受体脱敏和内化背后的机制，旨在开始揭示耐受性背后的分子机制。受体的脱敏和内化需要蛋白激酶和抑制蛋白的相互作用。该项目的目的是确定激酶和抑制蛋白在 mu-阿片受体脱敏和内化过程中的激动剂依赖性特异性。这项研究将利用已删除假定磷酸化位点的 mu-阿片受体的野生型和突变体。这些受体将用荧光蛋白标记并转染到组织培养细胞系中。将开发针对受体磷酸化形式的特异性抗体，并将其用于蛋白质印迹分析和免疫细胞化学。荧光共振能量转移（FRET）将用于确定受体和辅助蛋白的时间相互作用，并使用流式细胞术和共聚焦成像进行定量。