This 5 year renewal application is directed at using 14Beta- bromoacetamidomorphine (BAM) and 14Beta-bromoacetamidomorphinone (BAMO) to affinity label and purify the Mu opioid receptor. In the absence of a disulfide bond reducing agent, BAM and BAMO bound reversibly to opioid binding sites in rat brain membranes. However, after reduction of a disulfide bond at or near the Mu opioid binding site, BAM and BAMO bound specifically and covalently to this site. Greater than 90% of Mu opioid binding was inhibited, while Delta and Kappa binding were not affected by B M and BAMO alkylation of membranes. Protection experiments using opioids specific for the different types of opioid receptors, will determine the specificity of the alkylation and characterize the binding site that is alkylated with BAM and BAMO. The affinity and specificity of the reversibl and irreversible binding of [3H]BAM and [3H]BAMO to rat and bovine brain membranes will be determined. The molecular weight of the protein(s) label d with [3H]BAM and [3H]BAMO will be determined by separating brain membranes and partially purified opioid receptor complexes on SDS polyacrylamide gels followed by gel slicing or autoradiography. Proteins specifically labeled with [3H]BAM and [3H]BAMO will be purified under denaturing conditions, and a partial amino acid sequence of the protein(s) will be determined after proteolytic digestion of the protein(s). Studies will also be directed at improving solubilization procedures for obtaining active opioid receptors from rat and bovine brain membranes. Solubilization protocols and binding assay methodology will be optimized so that the binding of a number of radiolabeled opioid alkaloids and peptides to solubilized receptors can be measured reliably. After having improved the solubilization of active opio d receptors, affinity chromatography with BAM and BAMO will be used to purify active Mu opioid receptors. Partially purified opioid receptors will be labeled with [3H]BAM and [3H]BAMO to identify the opioid binding site. The second part of this proposal is directed at determining the role that prote n kinase C (PKC) may play in the downregulation of opioid receptors in NG108- 15 cells. In the presence of an opioid agonist, activiation of PKC by a phorbol ester enhanced the downregulation of opioid binding sites in comparison to cells treated only with an agonist. Studies are directed at determining the specificity and mechanisms by which PKC activation stimulat s opioid-induced downregulation in NG108-15 cells. Second messenger effects such as adenylate cyclase and the Na+/H+ antiporter exchange will be studie to determine if PKC is exerting its effect through either of these second messenger systems.
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