The goal of the proposed research is the purification and characterization of opiate receptors of neuronal tumor cell lines. Opiate receptors are membrane-associated proteins which provide the sites of action of opiates and of a family of structurally-related opioid peptides, possibly neuro-transmitters or hormones involved in nervous system pathways including that of pain perception. The NCB-20 cell line is a mouse neuroblastoma x Chinese hamster brain clonal hybrid which bears Delta, K, and Sigma opiate receptors. Purification of these receptors will involve affinity chromatography, ion-exchange chromatography, and ultimately the use of monoclonal antibodies directed against the receptor substypes. Characterization of the solubilized receptors will emphasize the structural features of the opiate binding site, and receptor subtype size, subunit composition, conformation, and mechanisms of action. Standard hybridization methods will be used in an attempt to isolate monoclonal antibodies directed against these receptors. Monoclonal antibodies have the potential to be excellent probes for the opiate receptor, because is should be possible to produce ones that are specific for specific determinants on the receptor surface. The monoclonal antibodies will be used in 1) affinity procedures for further purification of neural tumor cell opiate receptors, 2) immunohistochemical procedures to localize opiate receptors of the CNS and 3) determination of regions of structural homology among the receptor subtypes. Such studies are expected to help elucidate the molecular basis of opiate receptor heterogeneity. It is hoped that characterization of the opiated receptor subtypes will prove useful as biochemical markers of neural neoplastic transformation.
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