This is a Competing Continuation of a PPG with major themes of regulation of epithelial transport and differentiation. Four Projects supported by 3 Cores are proposed. The major areas of study are I) structure/function with emphasis on protein kinase regulation of epithelial transport proteins and 2) regulation of gene expression for several epithelial transport proteins, specifically induction by hypertonicity and by differentiation. All proposed projects involve transport proteins, which are enriched in epithelial cells and have been cloned by PI's of the PPG. The program represents a comprehensive, multi-disciplinary program centered in the JHU GI Division, with components in the Renal Division and Departments of Physiology and Biochemistry. A major strength of this program is the sharing of sophisticated molecular and cell biologic approaches among Projects. Project I: will increase understanding of how the brush border Na/H exchanger NHE3 functions at a molecular level; including probing the meaning of Vmax changes in kinase regulation, understanding the role of phosphorylation in its regulation in a polarized intestinal epithelial cell, the role of calmodulin as an inhibitor, how the membrane spanning domain and cytoplasmic domain of the molecule interact to bring about Vmax regulation, and NHE3 membrane topology. 2) Project 2: will increase understanding of how the renal osmolyte transporter, betaine/GABA transporter (BGT1) is regulated by purifying and then cloning a transcription factor called TonE binding protein; which binds to the BGT1 promoter and mediates the hypertonicity-induced transcriptional stimulation; studying regulation of TonEBP by hypertonicity, including post-translational regulation of BGT1 by protein kinases A and C. 3) Project 3A: will study two related NHE3 associated regulatory proteins, called E3KARP and NHE regulatory factor which are necessary for cAMP regulation of NHE3. Their role in NHE3 phosphorylation, subcellular distribution in intestinal cells, and which of their domains interact with NHE3 will be studied. The Cores are a Cell Culture Core; and an expanded Fluorescence Core, which now includes a confocal microscope, computerized fluorometers, fluorescence microscope/imaging system, and a luminometer.
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