Molecular Mechanisms of mu-Opioid Receptor Processing

描述(由申请人提供)： 阿片受体是吗啡及其衍生物等止痛药的主要作用部位。这些药物是有效的止痛药，尽管它们会产生成瘾和耐受性，使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，以期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阻滞素的相互作用。该项目的目的是确定激动剂依赖的特异性的激酶和arrestin作用于阿片受体的脱敏和内化。这项研究将利用野生型和突变型的Mu-阿片受体，其假定的磷酸化位点被删除。这些受体将被标记有荧光蛋白，并被导入组织培养细胞系。针对该受体磷酸化形式的特异性抗体将被开发出来，并用于蛋白质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，并将使用流式细胞仪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.