In order to answer one of the many questions on the multiple opioid receptors, i.e. whether or not these receptors represent different gene products, different splicing of the same gene, or post- transnational modification of the products, it is the goal of the current proposal to clone for the kappa-opioid receptor. Kappa- opioid receptor will be cloned from either the guinea pig cerebellum or human placenta library by the cDNA expression method. The library will be enriched in kappa-opioid receptor clones with 3 sets of probes: (a) restriction enzyme fragments of the mu- and delta-opioid receptor clones; (b) subtraction probes synthesized from hybridizing mRNAs from tissues containing low level of kappa- opioid receptor from that containing high level of kappa-opioid receptor, i.e. chronic kappa agonist (U50-488) or antagonist (MR2266) treatment will be used to alter the kappa-opioid receptor level and hence mRNA levels; and (c) the oligodeoxynucleotides sequences of the putative transmembrane regions V, VI and VII of the cloned 8-adrenergic receptor. The cDNA clones which hybridize with the first two sets of probes or with all three sets of probes will be expressed in eukaryotes previously devoid of opioid receptor activities. Clones which induced kappa-opioid receptor binding activity will be subcloned into Sp6 vectors for sense and anti-sense RNA synthesis. The RNAs thus synthesized will be injected into frog oocytes and the ability of kappa agonist to regulate the Ca+2 channels will be used to substantiate the identity of the clones. Antibodies will be developed against the deduced peptide sequence and will be used to immunoprecipitate ligand-kappa-opioid receptor complex and/or used to inhibit the kappa-opioid receptor binding activities in brain membranes. Such antibodies will be used also in immunocytochemical analysis of kappa-opioid receptor distribution and compared with the reported distribution of putative kappa binding sites. The identity of the kappa-opioid receptor clone will be substantiated further by the isolation and sequencing of the 125I-B-endorphin-receptor complexes when labelling was carried out in the presence of mu- and delta- opioid ligands. The gene structure and the nucleotide sequence of the kappa-opioid receptor clone will be determined and compared with that of mu- and delta-opioid receptor clones. Deletion and nucleotide insertion mutation studies will be carried out to investigate the structural requirement for kappa-opioid receptor activities.
