REGULATION OF THE NA+/K+ 2C1 COTRANSPORTER PROTEIN

这项提案的重点是对哺乳动物布美他尼的监管- 在粗大的升肢中发现敏感的Na-K-2Cl协转运体(RBSC) 亨勒的。这种蛋白是环状利尿剂的药理受体。 (速尿、布美他尼和乙丙氨酸)，并在 肾脏生理学。赞助商的实验室最近克隆了 测定了电子中性体Na-(K)-Cl的前三个成员的序列 辅转运蛋白家族，包括RBSC。这项提案的第一阶段涉及 与rRBC共转运蛋白的直接磷酸化作用 在调节其活性和离子化学计量方面。抗体 将生成rbsc的独特细胞质片段，如 重组融合蛋白。这些抗体将被用来 鉴定rbsc蛋白，并证明它是一种磷酸蛋白。 放射性标记RBSC的双向电泳和放射自显影 多肽片段将用于鉴定被PKA磷酸化的残基 和PKC。此外，还将通过高效液相色谱分离纯化并进行测序。 该方案确定的磷酸化位点将被改变的位点- Rbsc基因的定向突变。野生型和野生型稳定的转基因株 在缺乏内源性的细胞系中，将产生类型和突变的rbsc Na-K-2Cl共转运。这些已定义的突变的影响将是 通过离子共转运的功能分析进行评估。 BSC基因的转录调控是第二阶段研究的重点 这笔赠款。PKA和PKC在BSC mRNA诱导中的作用 在Northern印迹实验和核径流分析中进行评估，使用 大鼠(RBSC)和小鼠(MBSC)的cDNA探针。小鼠的BSC基因将是 从基因组文库中克隆的。它的内含子-外显子结构将是 确定，5‘-启动子区域将被克隆和测序。 MBSC启动子的功能特征将定义 组织特异性表达和反应所需的元件 致PKC。与mBSC相互作用的核蛋白的特性 赞助商将在授权期接近尾声时开始。长期的 第二阶段的目标是鉴定和克隆转录 调节BSC基因表达的因子。以…为类比 在其他组织中，这些转录因子(S)被认为起作用 在粗大的上肢的发育、生长和功能中 亨勒。

The focus of this proposal is the regulation of the mammalian bumetanide- sensitive Na-K-2Cl cotransporter (rBSC) found in the thick ascending limb of Henle. This protein is the pharmacological receptor for loop diuretics (furosemide, bumetanide and ethacrynic acid), and plays a pivotal role in renal physiology. The sponsor's laboratory has recently cloned and sequenced the first three members of the electroneutral Na-(K)-Cl cotransporter family, which includes rBSC. Phase I of this proposal deals with the role of direct phosphorylation of the rRBC cotransporter protein in the regulation of its activity and ionic stoichiometry. Antibodies will be generated to unique cytoplasmic fragments of rBSC, as expressed in recombinant fusion proteins. These antibodies will be used to characterize the rBSC protein and demonstrate that it is a phosphoprotein. Two dimensional electrophoresis and autoradiography of radiolabeled rBSC peptide fragments will be used to identify residues phosphorylated by PKA and PKC. Phosphopeptides will also be purified by HPLC and sequenced. Phosphorylation sites identified by this protocol will be altered by site- directed mutagenesis of the rBSC cDNA. Stable transfectants of both wild type and mutant rBSC will be created, in cell lines which lack endogenous Na-K-2Cl cotransport. The effect of these defined mutations will be assessed by functional assay of ion cotransport. The transcriptional regulation of the BSC gene is the focus of Phase II of this grant. The effect of PKA and PKC on induction of BSC mRNA will be assessed in Northern blot experiments and nuclear run-off assays, using the rat (rBSC) and mouse (mBSC) cDNA probes. The mouse BSC gene will be cloned from a genomic library. Its intron-exon structure will be determined, and the 5'-promoter region will be cloned and sequenced. Functional characterization of the mBSC promoter will define which elements are required for tissue-specific expression and for the response to PKC. Characterization of nuclear proteins which interact with the mBSC promoter will begin towards the end of the grant period. The long-term goal of Phase II is the identification and cloning of transcription factors which regulate the expression of the BSC gene. By analogy with other tissues, these transcription factor(s) are postulated to play a role in the development, growth and function of the thick ascending limb of Henle.