Little is known about the regulation of gap junction assembly and even less is known about gap junction removal from the cell surface. Such information is critical to the understanding of gap junctions and the role of hormonal control of cell-cell communication. In this proposal, the hypothesis that gap junction function may be regulated by the assembly and removal of gap junction protein from the plasma membrane will be tested. It is proposed that gap junction protein assembly and degradation are regulated by peptide hormone stimulation. Specifically, the role of adrenocorticotropin (ACTH) and cyclic adenosine monophosphate (cAMP) in regulating connexin 43 (Cx43) gap junction protein intracellular movement, channel assembly, and degradation will be studied in adrenal cells. Adrenal cortical cells that have gap junctions (SBAC and SW13 cell cultures) and those that lack gap junctions (H295 cells) will be transfected to express cDNA encoding for green, red or yellow protein Cx43. Gap junction protein processing (synthesis, trafficking, degradation and function) will be characterized by immunocytochemistry, live-cell imaging, western blot analysis and Lucifer yellow dye transfer. The spacial and temporal distribution of the Cx43 gap junction protein in secretory pathway organelles and in the plasma membrane will be determined in treated and nontreated cultures. Analysis of gap junction protein processing is an essential step in understanding the dynamics of gap junction and peptide hormone interaction. More importantly, this work will have a broad impact on the field of cell biology since it will provide a clearer understanding of the mechanisms by which hormones regulate organ function. In addition to the scientific impact, this study will provide a training environment for high school students, undergraduates, graduates, and postdoctoral fellows.
