MOLECULAR ASPECTS OF THE MULTIPLE OPIOID RECEPTORS

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This proposal is directed at using monoclonal antibodies directed against the opioid receptor to elucidate the molecular and physiological basis of the multiple opioid receptors. A monoclonal antibody, OR-689.2.4, capable of inhibiting opioid binding to neural membranes and precipitating the receptor from a solubilized preparation, has been obtained. A procedure has been developed for obtaining Fab fragments from this IgM. The Fab fragments will be studied for their ability to inhibit opioid binding to the different types of opioid receptors in neural membranes, cell lines, peripheral tissue, and solubilized receptor preparations. The effect of Na+ and GTP on the ability of the antibody to inhibit opioid binding will be studied to determine if the antibody modifies the inhibition of opioid binding caused by Na+ and GTP, or if Na+ and GTP alter the antibody's ability to inhibit opioid binding. These studies may further elucidate the mechanism by which Na+ and GTP inhibit opioid agonist binding but not antagonist binding. To determine if the antibody can alter an opioid-mediated physiological response, the Fab fragments will be tested to ascertain if they are capable of modulating the opioid-mediated inhibition of adenylate cyclase activity in the NG108-15 cell line and neural membranes. The antibody will also be tested for its ability to modify analgesia, using the tail flick and writhing assays. These studies may determine whether the antibody is acting as an agonist or antagonist. The Fab fragments from OR-689.2.4, covalently conjugated to Sepharose to form an affinity column, will be used to purify the opioid receptor. Specifically eluted material from the antibody affinity column will be assayed for its molecular characteristics and its ability to bind opioid ligands. Finally, additional monoclonal antibodies will be generated, using the material eluted from the antibody affinity column and the partially purified receptor preparation as antigen. The antibody will be provided to collaborators to use in studies ranging from molecular biology to whole animal assays.