PLCgamma regulates HNE3 activity through direct binding and dynamic complexes

描述（由申请人提供）： 在胃肠道中，水和营养物质的吸收作为消化的正常部分发生。在肠道中，钠吸收受损导致水分吸收减少和腹泻。在消化道疾病中，神经体液物质和细菌毒素导致细胞内第二信使（如钙（Ca2+））升高，导致肠和结肠中钠吸收减少。钠/氢交换器（NHE）基因家族在哺乳动物肠道钠吸收中起着不可或缺的作用。刷状缘NHE 3的调节解释了在消化期间发生的钠吸收的大多数公认的消化变化以及在腹泻期间发生的钠吸收的抑制。细胞内Ca 2+对NHE3活性的抑制可能涉及磷脂酶C γ（PLC g）的参与，但对Ca 2+调节NHE3活性的机制还不清楚。生物信息学分析表明，PLCg可能通过直接的蛋白质-蛋白质相互作用调节NHE3，并导致分子间PH结构域的形成。因此，本研究旨在验证以下假设：（1）NHE3和PLCg直接相互作用;（2）PLCg有助于基础活性，并且是Ca 2+抑制NHE3活性所必需的;（3）PLCg将NHE3支架到信号蛋白，如c-Src，其是NHE3活性的适当功能和调节所必需的;（4）NHE3和PLC g结合导致形成分子间PH结构域，其通过结合肠上皮细胞的脂筏微结构域中的特异性磷脂来调节NHE3活性。为了回答这些问题，主要研究者将利用体外结合试验（例如，下拉、酵母双杂交），通过诱变确定NHE 3中负责与PLC g直接结合的氨基酸。研究者将确定阻止PLC g与NHE3结合对NHE3活性的基础和Ca 2+抑制的后果。将进行生化研究，包括蔗糖密度梯度离心，以分析PLC g对NHE 3复合物形成的贡献。最后，研究人员将进行脂质结合试验，以发现磷脂和NHE 3之间的新相互作用，如生物信息学分析所预测的那样。这项研究的结果将提供新的见解PLCg在调节NHE3活性的作用，以进一步了解钠吸收消化和消化道疾病。 公共卫生相关性：在小肠和结肠中，NHE 3活性的调节解释了在消化期间发生的钠吸收的大多数公认变化以及在腹泻期间发生的钠吸收的抑制。细胞内钙对NHE 3活性的抑制可能涉及PLC g的参与，然而，负责调节的机制尚不清楚。目前的研究将为了解PLCg在消化和腹泻中调节NHE3活性的作用提供新的见解。

DESCRIPTION (provided by applicant): In the gastrointestinal tract, the absorption of water and nutrients occurs as normal part of digestion. In the intestine, impaired sodium absorption results in decreased water absorption and diarrhea. In diarrheal diseases, elevation of intracellular second messengers, such as calcium (Ca2+), by neurohumoral substances and bacterial toxins results in decreased sodium absorption in the intestine and colon. The sodium/hydrogen exchanger (NHE) gene family plays an integral role in sodium absorption in the mammalian intestine. Regulation of brush border NHE3 accounts for most of the recognized digestive changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs during diarrhea. Inhibition of NHE3 activity by intracellular Ca2+ may involve the participation of phospholipase C gamma (PLCg), however the mechanism responsible for Ca2+ regulation of NHE3 activity is not well understood. Bioinformatics analysis has suggested PLCg may regulate NHE3 through a direct protein-protein interaction and results in the formation of an intermolecular PH domain. Therefore, the current study is designed to test the hypothesis that (1) NHE3 and PLCg directly interact; (2) PLCg contributes to basal activity and is necessary forCa2+ inhibition of NHE3 activity; (3) PLCg scaffolds NHE3 to signaling proteins, such as c-Src, which are required for proper function and regulation of NHE3 activity; (4) NHE3 and PLCg binding results in the formation of an intermolecular PH domain which regulates NHE3 activity by binding specific phospholipids in lipid raft microdomains of intestinal epithelial cells. To answer these questions, the principal investigator will utilize in vitro binding assays(e.g. pull-down, yeast two-hybrid) to determine the amino acids within NHE3, by mutagenesis, that are responsible for direct binding to PLCg. Investigator will determine the consequence of preventing PLCg binding to NHE3 on basal andCa2+ inhibition of NHE3 activity. Biochemical studies, including sucrose density gradient centrifugation, will be performed to analyze the contribution of PLCg on formation of NHE3 complexes. Finally, the investigator will perform lipid binding assays to uncover novel interactions between phospholipids and NHE3 as predicted by bioinformatics analysis. The results of this study will provide novel insights into the role of PLCg in regulation of NHE3 activity to further understand sodium absorption in digestion and diarrheal diseases. PUBLIC HEALTH RELEVANCE: In the small intestine and colon, regulation of NHE3 activity accounts for most of the recognized changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs during diarrhea. Inhibition of NHE3 activity by intracellular calcium may involve the participation of PLCg, however, the mechansim [sic] responsible for the regulation is not well understood. The current study will provide novel insights into understanding the role of PLCg in regulating NHE3 activity in digestion and diarrhea.