We propose to further understand how the cloned mammalian brush border Na/H exchanger isoform NHE3 functions at a molecular level. Concentration will be on mechanisms by which protein kinases and growth factors regulate NHE3, acting by its C- terminus. Reasons for concentrating on NHE3 is that it appears to be the physiologically important brush border isoform, being involved in Na absorption by the intestine between meals and in the post-prandial increase in ileal Na absorption. A major area of emphasis is the kinetics by which growth factors/protein kinases regulate NHE3; it is regulated by changes in Vmax. In contrast, all regulation of the housekeeping isoform NHE1, which is present on the intestinal basolateral membrane, is by changes I K~(H+). We will study 1) The meaning of the Vmax regulation of NHE3, asking whether this is due to changes in the number of functional NHE3 molecules on the plasm membrane, a change in the turnover number of each exchanger, or both. Quantitation of amount of plasma membrane NHE3 will be via cell-surface biotinylation and quantitative Western analysis using an NHE3 fusion protein as an internal standard. Studies will be of NHE3 expressed in PS120 fibroblasts and Caco-2 cells, a polarized intestinal cell line. 2) The role of NHE3 phosphorylation in growth factor/kinase regulation of NHE3 expressed on the apical surface in polarized Caco-2 cells will be studied by in vivo phosphorylation using separation on one dimensional SDS-PAGE and auto-radiography, and two dimensional electrophoresis of tryptic digests of NHE3. 3) The mechanisms of NHE3 inhibition by calmodulin which occurs at basal Ca2+ will be probed examining calmodulin binding to NHE3 C-terminal fusion proteins and substitution of the NHE3 calmodulin binding domain with calmodulin binding domains from other proteins. 4) The NHE3 membrane spanning domain and cytoplasmic domain interact to define the kinetics of growth factor/kinase regulation. Membrane domain chimeras will be engineered to determine the region of the NHE3 membrane domain which is involved in the Vmax regulation. 5) To interpret the sits of interaction of the NHE3 membrane and cytosolic domains, increased understanding of NHE3 topology is required. Detailed study of the topology of NHE3 will be performed by two independent methods: (a) in vitro transcription/translation method in which putative membrane spanning domains (MSD) of NHE3 are synthesized in a cell free system as a fusion protein with an N-glycosylated tag to assess the location (intracellular or extracellular) of the fusion protein and (b) a N-glycosylation scanning mutagenesis approach.
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