Molecular mechanisms for chronic opioid actions has not been fully elucidated yet. Earlier reported studies, using clonal cell line models, suggested probable cellular modification of the receptor. Chronic administration of opioid agonists in vitro to tissue culture cells, such as neuroblastoma x glioma NG108-15, or in vivo to animals, have resulted in an alteration of the steady state level of opioid receptor. Previous efforts to determine how neuronal cells regulate opioid receptor level during chronic agonist exposure have been hampered by the lack of receptor reagents, such as receptor specific antibodies or the cDNA clone of the receptor. Such problems have been alleviated by recent reports of the cloning of three major types of opioid receptors, mu-, delta- and kappa- opioid receptors. We have since developed delta-opioid receptor specific polyclonal antibodies, using the peptide sequences specific for delta- opioid receptor and have designed delta-opioid receptor specific oligodeoxynucleotides that they have been used successfully in quantitating the level of the delta-opioid receptor messages in NG108-15 cells. Thus, with these reagents, we could now examine how neuronal cells, such as neuroblastoma x glioma cells control the steady state level of the receptor. The itinerary of the delta-opioid receptor in the presence of agonist will be examined. The turnover rate of the receptor protein will be determined. Cellular components which are involved in the endocytosis and trafficking of the receptor will be determined. In addition to tracking the receptor with receptor specific antibodies, we are also developing hemagglutinin epitope tagged delta-opioid receptor utilizing commercial available monoclonal antibodies to this epitope. The receptor domains involved in the receptor internalization and trafficking to lysozomes will be determined by mutational analysis of the intracellular domains of the receptor. Cellular components tightly associated with the receptor after agonist binding, such as the small GTP- binding proteins, will be identified by isolation of the agonist-receptor complexes using immunoaffinity columns. Regulation of the receptor protein level via transcriptional and translational control will also be examined. Cellular control of receptor transcription will be investigated by quantitating the delta-opioid receptor mRNAs under various conditions. Toward understanding the transcriptional regulation, the delta-opioid receptor gene will be isolated and characterized. The 5-upstream regulation elements will be identified. Cellular control of these regulation elements' activities will be investigated. By determining the mechanism of cellular regulation of both transcript and protein levels of delta-opioid receptor, we can then begin to understand neuronal cell regulation of the steady state level of the receptor and probable involvement of these cellular events in the expression of chronic opioid effects.
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