Molecular Mechanisms of mu-Opioid Receptor Processing

描述（由申请人提供）：

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.