Five types of opioid receptors have been demonstrated by bioassays on isolated tissues and binding studies on brain membranes. The molecular basis of the receptor heterogeneity is not known. The availability of an irreversible mu antagonist, beta-FNA, makes the mu receptor amenable to isolation and structure elucidation. The applicant's long-term objectives are to delineate the complete amino acid sequence of the mu opioid receptor, to characterize the functional domains of the mu receptor such as the ligand binding site and the coupling site for the second messenger and to study at the molecular level the biochemical mechanisms of development of opioid tolerance and dependence. The hypothesis of this grant application is that by using (3H)beta- FNA it will be possible to isolate the mu opioid receptor and determine partial amino acid sequence of its binding domain.
The specific aims of the proposed research are: (1) covalent labeling of the mu opioid receptor with (3H) beta-funaltrexamine ((3H)beta-FNA), (2) purification of the pre-labeled receptor by use of affinity chromatography and preparative gel electrophoresis, and determination of its amino acid composition (3)determination of the partial amino acid sequence of its binding domain by limited proteolysis of the labeled receptor followed by purification with high performance liquid chromatography and gas-phase sequencing, (4) generation of antibodies against peptides synthesized according to the partial sequence determined, and (5) characterization of the receptor by use of antibodies, such as immunoprecipitation of receptor binding activities and immunohistochemistry. Molecular cloning of the receptor by use of synthetic oligonucleotide probes coded for the peptide sequence or antibodies against the receptor can thus proceed to delineate the complete amino acid sequence of the receptor. The complete amino acid sequence allows prediction of its secondary structure and functional domains. The labeled fragment determines the binding domain of the receptor. Site- directed mutagenesis allows determination of the coupling site to the second messenger. Comparison of the amino acid sequence with those of other types of opioid receptors (such the delta receptor) will provide information on the molecular basis of receptor heterogeneity, and help in the design of selective drugs for each receptor. The proposed research will lead to studies on regulation of expression of the mu receptor at the molecular level, which will shed light on the mechanism of development of opioid tolerance and dependence.
