Opioid receptors play important roles in many physiological functions. At least four types of opioid receptors (mu, delta, kappa, epsilon) have been demonstrated by bioassays on isolated tissues and binding studies on brain membranes. The molecular basis of the receptor heterogeneity is now known. The availability of an irreversible mu antagonist, beta- funaltrexamine (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, and to study at the molecular level the biochemical mechanisms of development of opioid tolerance and dependence. The hypothesis of this application is that by using [3H] beta-FNA as an affinity label it will be possible to purify labeled fragments of the mu opioid receptor, to determine partial amino acid sequence of the receptor, to delineate the entire amino acid sequence by molecular cloning and to generate antibodies against fragments of the binding domain.
The specific aims of the proposed research are: (1) To purify the [3H] beta-FNA-labeled fragments of the mu receptor from bovine striatal membranes and to determine partial amino acid sequences of these fragments by limited proteolysis of partially purified receptors followed by purification by immunoaffinity column with antibodies recognizing beta-FNA and HPLC; (2) To clone the bovine mu opioid receptor by screening bovine brain or striatum cDNA libraries with oligonucleotide probes synthesized according to the amino acid sequence; (3) To generate antibodies against peptides synthesized according to the amino acid sequence for characterization of the receptor by immunohistochemistry, inhibition of opioid binding to membranes, immunoprecipitation of receptor binding activities. The delineation of the entire amino acid sequence allows prediction of its secondary structure and functional domains. The labeled fragment determines the binding domain of the receptor. Comparison of the amino acid sequence with those of other types of opioid receptors 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.
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