Molecular Mechanisms of mu-Opioid Receptor Processing

描述（由申请人提供）： 阿片受体是镇痛药物如吗啡及其衍生物的主要作用部位。这些药物是有效的镇痛药，尽管它们可能产生成瘾和耐受性，使其临床使用存在问题。本研究的基本原理是了解阿片受体脱敏和内化背后的机制，目的是开始解开耐受背后的分子机制。受体的脱敏和内化需要蛋白激酶和抑制蛋白的相互作用。本项目的目的是确定激动剂依赖的特异性激酶和抑制蛋白的作用在脱敏和内化的μ阿片受体。本研究将利用μ-阿片受体的野生型和突变体，其缺失了推定的磷酸化位点。这些受体将用荧光蛋白标记并转染到组织培养细胞系中。将开发特异于受体磷酸化形式的抗体，并用于Western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，并将使用流式细胞术和共聚焦成像进行定量。

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.