The direct metabolic and electrical coupling of cells by means of gap junctions has far significance for the integration of tissue function as a whole and specific relevance to the regulation of cell proliferation. Tumor and transformed cells typically show reduced coupling and many transforming agents (eg. oncogenes, tumor promotors) seem to down-regulate junctional expression. The reverse has also been reported for some anti-neoplastic agents (eg. retinoic acid). Clones (cDNA) to several of the known gap junction proteins have now been isolated and clearly show there to be a family of related proteins showing major differences in their regulatory domains. As a first step, the gap junctional channel structure and locations of functional domains on the protein subunits will be studied and compared between the various subtypes. Initial models will be tested and modified using results from in vitro manipulations (eg. phosphorylation), peptide analyses, and site specific antibodies. Functional effects on the junctions will be monitored electrophysiologically in isolated preparations employing the """"""""tip- patch"""""""" technique, or in situ in cultured cells. Refinement of these models will be achieved by expression of the proteins in frog oocytes, and subsequently testing the effects of oligonucleotide directed mutations on intercellular channel function. These studies will serve to better describe channel structure, selectivity (if found in the various subtypes) and modes of regulation. Parallel experiments will use conserved and variable region probes from the junctional cDNA's to determine the approximate size of the gene family and the patterns of expression of the different junction subtypes before and after the transformation process, including their distribution within selected tumors. Finally, the two lines of investigation will be combined by transforming cells with expression vectors containing the junctional cDNA's (in both sense and antisense orientations) and directly testing the necessity or sufficiency of junctional downregulation in the process of transformation and metastatic invasion of issue.
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