Hormone Regulation of Gap Junction Processing

对缝隙连接组装的调控知之甚少，对细胞表面缝隙连接的去除更是知之甚少。这些信息对于理解缝隙连接和荷尔蒙控制细胞-细胞通讯的作用至关重要。在这一提议中，将检验缝隙连接蛋白可能通过从质膜组装和移除缝隙连接蛋白来调节缝隙连接功能的假设。有人认为缝隙连接蛋白的组装和降解受肽激素刺激的调节。具体地说，促肾上腺皮质激素(ACTH)和环磷酸腺苷(CAMP)在调节连接蛋白43(Cx43)缝隙连接蛋白在肾上腺细胞内的运动、通道组装和降解中的作用将被研究。有缝隙连接的肾上腺皮质细胞(SBAC和SW13细胞培养)和没有缝隙连接的肾上腺皮质细胞(H295细胞)将被导入表达编码绿色、红色或黄色蛋白Cx43的cDNA。缝隙连接蛋白的加工(合成、运输、降解和功能)将通过免疫细胞化学、活细胞成像、蛋白质印迹分析和荧光黄转移来表征。Cx43缝隙连接蛋白在分泌途径细胞器和质膜中的时空分布将在处理和未处理的培养物中确定。分析缝隙连接蛋白的加工过程是了解缝隙连接和多肽激素相互作用动力学的关键步骤。更重要的是，这项工作将对细胞生物学领域产生广泛的影响，因为它将提供对激素调节器官功能的机制的更清晰的理解。除了科学上的影响，这项研究还将为高中生、本科生、毕业生和博士后研究员提供一个培训环境。

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.