REGULATION OF THE RENAL BETAINE TRANSPORTER

甜菜碱在保护细胞免受高渗中起重要作用 应力肾髓质通常是高渗的， 高渗性根据水合状态而变化。 在一些 病理状态所有组织经历高渗并适应 不利的环境使用类似的机制， 肾髓质甜菜碱转运蛋白的调节在 甜菜碱在肾上皮细胞中的保护性积累。 当细胞暴露于高渗环境时， 编码甜菜碱转运蛋白的基因 刺激导致甜菜碱转运蛋白活性增加 和甜菜碱积累。 甜菜碱转运蛋白也受到调节 通过蛋白激酶A和C的激活剂进行后处理。 在 为了了解细胞如何感知高渗压力， 信号被转导到转录机器， 一种转录因子，介导转录刺激， 甜菜碱转运蛋白基因响应高渗将被克隆。 使用酵母的遗传选择策略，亲和筛选，或 将使用从纯化的蛋白质获得的氨基酸序列 克隆转录因子的cDNA 抗体与 将基于cDNA序列产生转录因子。 cDNA和抗体探针将被用来研究如何 转录因子被高渗激活。 如果调控 处于通过北方印迹确定的转录水平，并且 核连续试验，转录调控机制 将通过克隆转录因子的基因进行研究， 寻找调控序列元件。 一级的监管 蛋白质丰度，亚细胞定位的变化， 还将使用抗体研究磷酸化。 学习 蛋白激酶如何抑制甜菜碱转运蛋白的活性， 蛋白激酶的激活对细胞内 甜菜碱的磷酸化和亚细胞定位 将使用特异性抗体研究转运蛋白。

Betaine plays an important role in protecting cells from hypertonic stress. The renal medulla is normally hypertonic and the degree of hypertonicity changes depending on hydration status. In some pathologic states all tissues experience hypertonicity and adapt to the adverse environment using mechanisms similar to those of the renal medulla. Regulation of the betaine transporter is critical in osmo-protective accumulation of betaine in renal epithelial cells. When cells are exposed to a hypertonic environment, transcription of the gene coding for the betaine transporter is markedly stimulated resulting in increased activity of the betaine transporter and betaine accumulation. The betaine transporter is also regulated post-translationally by activators of protein kinases A and C. In order to understand how cells sense hypertonic stress and how the signal is transduced to the transcriptional machinery, the transcription factor that mediates transcriptional stimulation of the betaine transporter gene in response to hypertonicity will be cloned. A genetic selection strategy using yeast, an affinity screening, or amino acid sequence obtained from the purified protein will be used to clone the cDNA for the transcription factor. Antibodies to the transcription factor will be raised based on the cDNA sequence. The cDNA and antibody probes will be used to study how the transcription factor is activated by hypertonicity. If the regulation is at the level of transcription as determined by Northern blot and nuclear run-on assays, the mechanism of transcriptional regulation will be studied by cloning the gene for the transcription factor and searching for regulatory sequence elements. Regulation at the level of protein abundance, shift in subcellular localization, and phosphorylation will be also studied using the antibodies. To learn how protein kinases inhibit activity of the betaine transporter, the effects of activation of the protein kinases on changes in phosphorylation and subcellular localization of the betaine transporter will be studied using specific antibodies.