The opioid receptors belong to the family G protein coupled receptors (GPCRs), the largest family of membrane proteins in the human genome. Opioid receptor agonists are widely used for the management of pain. However, these drugs are also highly addictive and their clinical efficacy is often limited by the development of tolerance. There is a growing body of evidence that opioid receptors and other GPCRs are conformationally complex molecules. Structurally different agonists may induce functionally distinct receptor conformational states that differ in their interactions with G proteins, kinases, arrestins and possibly other signaling molecules. Therefore, the agonist-specific conformational state will determine the clinical efficacy of a drug. In addition to their clinical importance, opioid receptors are particularly valuable as a model system for understanding the mechanism of GPCR activation because of the broad spectrum of opioid ligands including peptide and small molecular weight agonists and antagonists. A better understanding of the opioid receptor structure and drug-induced conformational changes will impact efforts to develop more effective drugs. The goal of this Stage I CEBRA proposal is to demonstrate the feasibility of using fluorescence spectroscopy to study ligand-induced conformational changes in the mu-opioid receptor (MOR) and the delta-opioid receptor (DOR). Experimental Aims include: 1. Develop methods for the production and purification of functional MORs and DORs. 2. Develop methods for the functional reconstitution of MOR and DOR in to phospholipid vesicles and characterize the effect of lipid composition on receptor function. 3. Site-specific labeling of purified opioid receptors with thiol-reactive probes. 4. Study ligand-induced conformational changes in MOR and DOR labeled with conformationally sensitive fluorescent probes. Successful completion of these aims will provide the preliminary data for a more comprehensive study of opioid receptor structure and conformational dynamics in a Stage II CEBRA project.
