Molecular mechanism of opioid tolerance and dependence is a subject under intense investigations. Models have been developed so as to facilitate the understanding of these opioid pharmacological effects. Neuroblastoma x glioma NG108-15 cells is one such model. Previous studies with this model have suggested that the homogeneous population of delta-opioid receptor in this cell line is under stringent cellular regulation. Chronic opioid agonist treatment resulted in a loss in receptor's responses due to receptor desensitization and receptor down-regulation. At least in receptor down-regulation parallel observations with other opioid receptor types were obtained in animals chronically administered opioid receptor selective agonists. Thus, understanding of molecular basis for receptor desensitization and down-regulation could illuminate the problem of tolerance. Previous efforts have been hampered by lack of opioid receptor reagents, such as receptor specific antibodies. Now with recent cloning of delta-opioid followed by mu- and kappa-opioid receptor, receptor specific antibodies could now be developed. Therefore, in current studies, we propose to develop to delta-opioid receptor specific antibodies by immunizing rabbits with peptides synthesized according to deduced primary sequence of cloned delta-opioid receptor, and immunizing rabbits with receptor proteins expressed in and purified from E. coli. Identities of antibodies produced will be established by comparing western analysis of membrane isolated from CHO cells, CHO stably transfected with delta-opioid receptor cDNAs, and NG108-15 cells. Immunocytochemistry will be performed with brain slices and these antibodies in order to utilize known delta-opioid receptor distribution to characterize these antibodies. The abilities of these antibodies to inhibit opioid receptor binding, to immunoprecipitate delta-opioid receptor will be established. The hypothesis of receptor phosphorylation as mechanisms for receptor desensitization will be investigated using these receptor specific antibodies to examine the phosphorylation states of receptor during agonist treatment. Delta-opioid receptor will be separated from other phosphoproteins using these antibodies. Abilities of known protein kinases to phosphorylate delta-opioid receptor will be examined. Degree phosphorylation and sites of phosphorylation will be examined by peptide mapping of immunoprecipitated or immunoaffinity purified receptors. Effect of receptor phosphorylation will be investigated also by mutation analysis of cloned delta-opioid receptor. Delta-opioid receptor clone will be mutated by site-directed mutagenesis or deletion mutagenesis, with the focus on serine and threonine moieties in the cytosolic domains of the receptor molecule. Effect of these mutations on phosphorylation, receptor desensitization and receptor down-regulation will be evaluated by transient expression in COS7 cells and stable transfection in CHO cells of wide type and mutant delta-opioid receptor. Through all mutation studies and phosphorylation studies, we will establish the role of phosphorylation in cellular adaptation to chronic presence of opioid agonists.
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