Neuroblastoma (NB) cell lines have served as models in the study of opioid receptor structure and function; however, the presence of Delta sites only limited their value as a general model of the opioid receptor system. Screening in my laboratory of several human NB cells revealed the presence of Mu sites on at least one line, MC-NB-1, while Delta sites were expressed in several lines. The present project proposes to establish a number of suitable cell lines that express all of the major opioid receptor types and possibly subtypes. This will be accomplished by screening more existing NB lines, subcloning lines of interest, and producing somatic human-rodent cell hybrids. The latter can serve to localize the opioid receptor genes to specific chromosomes and to obtain hybrids with faster growth rates or higher receptor yield. Further, NB cell lines will be differentiated into mature neurons by several agents in order to study the regulation of opiate receptor expression. Special emphasis of the project will be placed on developing quantitative assays for the opioid receptor molecules, including detection with biotinylated Beta-endorphin and monoclonal antibodies in conjunction with immunochemical techniques (fluorescence, enzyme labels). These assays will be applied to fluorescence flow cytometry to determine receptor expression of single cells under various conditions (cell cycle phases, down-regulation, differentiation). In conjunction with fluorescence resonance energy transfer (e.g. fluorescein-labeled Mu receptor as donors and rhodamine labeled Delta receptors as acceptors), one can measure the average distance among receptor sites on a molecular scale (less than 100 Angstrom). This approach will be used to address the question of any physical association between Mu and Delta sites that are expressed on the same cell. Another important aspect of the proposal involves the question as to which second messengers are affected by the opioid receptors. We will concentrate on cAMP metabolism, phosphatidyl inositol turnover, and catechol metabolism and secretion, as well as CA++ disposition. Further, cell lines with high receptor yield will serve to solubilize and purify the opioid receptor types. One subsequent goal will include the production of a panel of monoclonal antibodies with varying epitope specificities. Finally, the current project will serve to foster a series of collaborative studies that have been initiated to elucidate the molecular biology of the opioid receptor system.
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