This proposal deals with several aspects of signal transduction which are unique to epithelial tissues. Differences in the signaling pathways in the apical and basolateral membranes (BLM) of intestinal epithelial cells are explored which are initiated by BLM signals generated by EGF (acting by a tyrosine kinase receptor), and carbachol (acting by a G- protein linked receptor). Both regulate the ileal apical membrane neutral NaC1 absorptive process and its component brush border (BB) Na/H exchanger. NHE3 with EGF causing stimulation and carbachol inhibition. A major topic of this proposal deals with how signals generated from the BLM are focused on BB processes, in this case on BB Na/H exchange. We hypothesize that this is achieved by BB signaling complexes that involve the long cytoplasmic C-terminus of the transport protein being regulated, NHE3. We propose to build on our novel observation that there are BB signaling complexes which contain NHE3; the tyrosine kinases c-Src and c-Yes; two PIP2 utilizing enzymes, PI 3-kinase and PI- PLCgamma; and two microvillus cytoskeletal components, villin and ezrin with EGF and carbachol acting by different members of these complexes. The insights in signal transduction to be gained from these studies will provide new insights into digestive physiology and the pathobiology of diarrheal diseases, and will provide new approaches to treat diarrheal diseases. We propose to study parallel aspects of EGF stimulation and carbachol inhibition of BB Na/H exchange. The approaches will involve measuring active Na and C1 transport in ileal mucosal sheets measured with the Ussing chamber/voltage clamp technique, ilea BB vesicle Na/H exchange, and Na/H exchange in the colon cancer cell line Caco-2 stably transfected with NHE3. EGF: a) The mechanism by which PI3-kinase mediates the EGF increase in Na/H exchange will be examined by cell surface biotinylation to determine if the Vmax regulation is due to more molecules of NHE3 on the Caco-2 cell BB or change in NHE3 turnover number and which aspect is dependent on PI3-kinase. Constitutively active PI 3-kinase will allow testing of whether PI 3-kinase activation is sufficient to stimulate BB NHE3. b) The involvement of Src family tyr kinases in stimulation of BB NHE3 will be determined by biochemical studies and use of constitutively active Src family mutants in Caco-2/NHE3 cells. c) The role of the NHE3 C-terminus in its stimulation by EGF when expressed in Caco-2/NHE3 cells will be examined and compared with the formation of signaling complexes with the NHE3 C- terminus partially truncated. Carbachol/protein kinase C: a) The involvement of Src family tyr kinases in inhibition of BB NHE3 by carbachol will be determined by biochemical transport studies in ileum. b) Caco-2/NHE3 cells as a model of carbachol inhibition of NHE3 will be developed. c) The role of the NHE3 C-terminus in inhibition by carbachol/protein kinase C when expressed in Caco-2/NHE3 cells will be examined with C-terminal truncation mutations and point mutations with correlation of formation of signaling complexes and inhibition of NHE3.
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