Gap junctions are the plasma membrane specializations, present between most types of contacting cells, through which the passive movement of small molecules from one cell to another occurs (direct cell-cell communication). Many studies have indicated that cell growth, development and differentiation may be regulated by the passage of small molecules through these junctions. The long-term objective of this study is to understand the significance of gap junctional communication, as well as how it is established and regulated. The major focus of the proposed studies is to identify the covalent modifications, such as covalent derivatization, that the gap junction undergoes along the pathway from initial synthesis of gap junction polypeptides through the assembly of mature gap junctions. Particular emphasis will be placed on how these modifications function to permit assembly of junctions and their involvement in regulation of communication. The steps in this process will be analyzed in primary cultures of hepatocytes and myocardial cells, as well as in a cell line in which the terminal steps in assembly can be induced by incubation with cAMP. The potential interaction of gap junction proteins with other cellular polypeptides will be investigated. These experiments capitalize on properties of a bank of antibodies which we have prepared to currently identified gap junction proteins. These antibodies include polyclonal and monoclonal antibodies to the 27 kDa liver gap junction protein, monoclonal anti-peptide antibodies to the 27 kDa liver gap junction protein, and polyclonal anti-peptide antibodies to the liver 27- and 21-kDa proteins as well as to the heart 43 kDa protein. Additional antibodies, both polyclonal and monoclonal, with different specificities for these proteins and to other homologous, but distinct, gap junction proteins are currently being generated for use in this investigation. Topological studies, using both biochemical and immunochemical approaches, will be undertaken to further develop models for the disposition of these proteins in the plasma membrane lipid bilayer, as well as to identify specific amino acid residues whose covalent modifications, such as phosphorylation by protein kinases, can regulate channel function. Reconstitution studies will be carried out in order to correlate in vivo modifications with channel activities measured using purified components. Through collaborative studies, the nature of central nervous system proteins that have shown immunoreactivity with our antibodies will be characterized, and gap junction assembly in uterus during gestation will be examined. Information obtained from these studies will be applied to analysis of other systems, including transformed cells in which gap junctional communication is altered or absent.
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