PLCgamma regulates HNE3 activity through direct binding and dynamic complexes

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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.