REGULATORY PROTEINS FOR CAMP DEPENDENT REGULATIONS OF NHE3

NHE3 的营地依赖性调节的调节蛋白

• 批准号: 6500424
• 负责人: Changhyon Chris Yun
• 金额: $ 10.37万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6500424
• 关键词: SDS polyacrylamide gel electrophoresis; binding proteins; cell membrane; cyclic AMP; fluorescence microscopy; gene expression; hydrogen; immunoprecipitation; in situ hybridization; ion transport; laboratory rabbit; membrane transport proteins; molecular biology; phosphorylation; protein kinase A; protein structure function; sodium potassium exchanging ATPase; transcription factor; transfection; western blottings; yeast two hybrid system

Cyclic AMP is a major second messenger playing an important role in electrolyte transport in intestinal and renal epithelial cells and contributing to pathophysiology of diarrheal diseases. An increased cAMP level in intestine is a major cause of diarrheal diseases primarily by increasing C1- secretion by crypt cells and by decreasing Na+ absorption by villus cells. The decrease in Na+ absorption in villus cells by cAMP is poorly understood except that inhibition of brush border Na+/H+ exchanger, NHE3, in enterocytes is a target. We were surprised that cAMP failed to regulate the brush border Na+/H+ exchanger, NHE3, in two model system, PS120 fibroblasts and Caco-2 cells. Using a yeast two-hybrid system, we have identified two regulatory proteins, E3KARP and NHERF, which are necessary for NHE3 inhibition by cAMP. These proteins show a limited homology but have a similar human tissue distribution. PS120 fibroblasts and human colonic carcinoma Caco-2 cell line lack endogenous expression of these regulatory proteins. When these regulatory proteins are expressed in PS120 fibroblasts stably transfected with NHE3, both regulatory proteins can reconstitute the cAMP-induced inhibition of NHE3, suggesting the necessity of the regulatory proteins. In order to understand the molecular basis for NHE3 inhabitation by cAMP, we propose to investigate interactions between NHE3 and the regulatory proteins at a molecular level. (1) We will investigate the effect of cAMP on changes in phosphorylation of NHE3 and the regulatory proteins in Caco-2 cells. (2) How the regulatory proteins transduce cAMP effects on NHE3 is not known. We hypothesize that the regulatory proteins may function as scaffold proteins for proteins kinase A, assembling components involved in cellular signaling at the plasma membrane. We will study interaction between PKA and the regulatory proteins. (3) We will raise antibodies against the regulatory proteins and determine subcellular localization of the regulatory proteins and NHE3. (4) If E3KARP and NHER are expressed in the same cells, we will investigate potential interactions between the two regulatory proteins such as dimerization and synergy in NHE3 inhibition by cAMP. (5) We will also determine the interaction domains within NHE3 and the regulatory proteins using a yeast two-hybrid system.

环磷酸腺苷是一种重要的第二信使， 在肠和肾上皮细胞的电解质转运中， 对牙周病的病理生理学有贡献。 增加 肠内cAMP水平是引起肠道疾病的主要原因 主要是通过增加隐窝细胞的C1分泌， 降低绒毛细胞对Na+的吸收。 Na+的减少 绒毛细胞对cAMP的吸收知之甚少， 抑制刷状缘Na+/H+交换器，NHE 3， 肠上皮细胞是一个目标。 我们很惊讶cAMP没有 调节刷状缘Na+/H+交换器，NHE 3，分为两部分 模型系统、PS120成纤维细胞和Caco-2细胞。 使用酵母 双杂交系统，我们已经确定了两个调控蛋白， E3 KARP和NHERF，它们是NHE 3抑制所必需的， 营 这些蛋白质显示出有限的同源性，但具有相似的 人体组织分布PS120成纤维细胞和人结肠 癌Caco-2细胞系缺乏这些内源性表达 调节蛋白 当这些调节蛋白表达时， 在用NHE 3稳定转染的PS120成纤维细胞中， 蛋白质可以重建cAMP诱导的NHE 3抑制， 提示调节蛋白的必要性。 为了 了解cAMP抑制NHE 3的分子基础， 我们建议研究NHE 3和 在分子水平上的调节蛋白。 (1)我们将调查 cAMP对NHE 3磷酸化变化的影响， Caco-2细胞中的调节蛋白。 (2)如何监管 cAMP对NHE 3的影响尚不清楚。 我们 我假设调节蛋白可以作为支架 蛋白激酶A的蛋白质，组装参与 细胞膜上的信号传导。 我们将研究 PKA与调节蛋白之间的相互作用。 (3)我们将 产生针对调节蛋白的抗体并确定 调节蛋白和NHE 3的亚细胞定位。 (4)如果 E3 KARP和NHER在相同的细胞中表达，我们将 研究两种调节蛋白之间的潜在相互作用 如cAMP抑制NHE 3的二聚化和协同作用。 (5)我们还将确定NHE 3内的相互作用域 和调节蛋白质。