Though opioid receptors were first identified almost 20 years ago, they still have not been purified. A major problem has been the difficulty in solubilizing them in a form which retains high ligand-binding activity and unaltered characteristics. One way around this problem is to covalently label the receptors prior to solubilization, for example, by cross-linking them to a peptide ligand such as beta-endorphin. Cross-linking technique has been successfully employed by several laboratories to identify polypeptides that bind beta-endorphin, but the twin problem of determining which these labelled bands represent bona fide receptors, and followed by the actual purification of these receptors remains. We propose to use a cross-linking technique to identify receptors for beta- endorphin, dynorphinA(1-13) and DADLE in several tissues, including rat periaqueductal gray (PAG), which is enriched in mu opioid receptors, NG108- 15 neuroblastoma x glioma hybrid cells, which contain a homogeneous population of delta receptors, and guinea pig cerebellum, which is enriched in kappa opioid receptors. To demonstrate the pharmacological relevance of cross-linked bands, we will apply several criteria, including selective competition by specific opioid ligands, coupling to G-proteins in the case of the PAG, and down-regulation by chronic agonist treatment in NG108-15 cells. Those bands that appear to be pharmacologically relevant will be purified using ligand-specific antibodies, such as anti-beta-endorphin-antibody, in conjunction with other procedures such as lectin affinity chromatography and HPLC. The purified proteins will be partially sequenced, oligonucleotides will be synthesized corresponding to these sequences, and used as probes to isolate cDNA from a rat brain library. We will further characterize the cross-linked bands by determining their levels in NG108-15 cell lines transfected with sense and antisense portions of the cDNA sequence that codes for OBCAM, an opioid binding protein purified from bovine brain. The antisense-transfected cells exhibit reduced 3H-diprenorphine binding, relative to non-transfected and sense- transfected NG108-15 cells; in contrast, muscarinic and alpha2-adrenergic binding in these cells do not differ from controls, suggesting the transfection has specifically affected opioid binding. We will also transfect COS-7 cells with OBCAM cDNA, and characterize these cells for opioid receptor binding, using both conventional receptor binding assays as well as cross-linking. Finally, we are in the process of creating a line of transgenic mice containing antisense OBCAM cDNA. Preliminary characterization of the first generation of mice suggests that their response to morphine may be altered. When breeding has continued through enough generations to establish a stable line of genetically altered mice, these animals will be thoroughly tested pharmacologically, including beta-endorphin cross-linking studies.
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