G Protein-Mediated Gene Regulation

描述（由申请人提供）：G蛋白偶联受体检测许多不同的外部信号，包括调节基因表达和细胞分化的信号。虽然G蛋白介导的信号转导途径的许多成分是已知的，但不同信号调节特定基因的机制仍有待确定。途径特异性，部分地，由G蛋白G（α）亚基，和最近的数据表明，这些亚基在土壤阿米巴Dictyosteelae的基因调控中发挥作用。这项研究的目的是验证G（α）亚基通过与其他信号成分（如MAP（丝裂原活化蛋白）激酶）相互作用来调节基因表达的假设。为了实现这一目标，将在缺乏MAP激酶Erk 2并表达不同野生型或嵌合G（α）亚基的细胞中检查盘状蛋白（Dsc）在网柄藻中的早期发育表达。此外，将使用遗传筛选来鉴定抑制突变，所述抑制突变从嵌合G（α）亚基的抑制中拯救pDSC/GFP报告基因表达。抑制突变将通过REMI诱变产生，允许标记可能在G（α）亚基下游发挥作用以调节基因表达的基因。将进行抑制基因的额外表征，以验证它们在这些信号传导途径中调节G（α）亚基下游DSC基因表达的功能。这些研究将为控制真核细胞分化的信号机制提供重要见解。

DESCRIPTION (provided by applicant): G protein-coupled receptors detect many different external signals including ones that regulate gene expression and cellular differentiation. While many components of G protein-mediated signal transduction pathways are known, the mechanisms by which different signals regulate specific genes remains to be determined. Pathway specificity is, in part, determined by the G protein G (alpha) subunits, and recent data indicate these subunits play a role in gene regulation in the soil amoebae Dictyostelium. The goal of the proposed research is to test the hypothesis that G (alpha) subunits regulate gene expression through interactions with other signaling components, such as MAP (mitogen-activated protein) kinases. To achieve this goal, the early developmental expression of discoidin (Dsc) in Dictyostelium will be examined in cells lacking the MAP kinase Erk2 and expressing different wild-type or chimeric G (alpha) subunits. In addition, a genetic screen will be used to identify suppressor mutations that rescue pDSC/GFP reporter gene expression from the repression of a chimeric G (alpha) subunit. The suppressor mutations will be created by REMI mutagenesis, allowing genes to be tagged that might function downstream of G (alpha) subunits to regulate gene expression. Additional characterization of suppressor genes will be conducted to verify their function regulating DSC gene expression downstream of G (alpha) subunits in these signaling pathways. These studies will provide significant insights into the signaling mechanisms that control the differentiation of eukaryotic cells.