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

该提案的重点是对哺乳动物布美他尼的监管- 在粗的上行支中发现了敏感的Na-K-2Cl协同转运蛋白（rBSC） 关于Henle 这种蛋白质是袢利尿剂的药理学受体 （呋塞米、布美他尼和依他尼酸），并在 肾脏生理学 赞助商的实验室最近克隆了 对电子中性Na-（K）-Cl的前三个成员进行了测序， 协同转运蛋白家族，包括rBSC。 本提案的第一阶段涉及 与rRBC协同转运蛋白的直接磷酸化作用 调节其活性和离子化学计量。 抗体 将产生rBSC的独特细胞质片段，如在 重组融合蛋白。 这些抗体将用于 表征rBSC蛋白并证明其为磷蛋白。 放射性标记rBSC的双向电泳和放射自显影 肽片段将用于鉴定PKA磷酸化的残基 和PKC。 还将通过HPLC纯化磷酸肽并测序。 本方案确定的磷酸化位点将根据位点- rBSC cDNA的定向诱变。 两种野生型的稳定转染子 型和突变型rBSC将在缺乏内源性的细胞系中产生， Na-K-2Cl共转运。 这些定义的突变的影响将是 通过离子共转运功能测定进行评估。 BSC基因的转录调控是第二阶段的重点， 这个grant。 PKA和PKC对BSC mRNA诱导的影响将被进一步研究。 在北方印迹实验和核径流测定中评估，使用 大鼠（rBSC）和小鼠（mBSC）cDNA探针。 小鼠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，并克隆和测序5 '启动子区域。 mBSC启动子的功能表征将确定 元件是组织特异性表达和应答所必需的 到PKC。 与mBSC相互作用的核蛋白的表征 发起人将开始对补助金期限结束。 长期 第二阶段的目标是鉴定和克隆转录本， 调节BSC基因表达的因子。 比照 在其他组织中，这些转录因子被假定在 在发育，生长和功能的厚上升肢体的 亨利

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.