In this amended PPG Application, 6 NIH-funded Pis propose interactive research focusing on the trafficking of membrane transport proteins in polarized epithelial cells from the small intestine, liver and kidney. Three Projects and 3 supporting Cores are proposed. The proteins, each of which contribute to human diseases, are the BB Na/H exchanger NHE3 (Project 1, PI: M. Donowitz), the intracellular Cl channel CIC-5 (Project 2, PI: S. Guggino, co-investigator W. Guggino), and the Cu-ATPases 7A (Menkes protein) and 7B (Wilson's protein) (Project 3, PI: A. Hubbard). The common questions being addressed are: What are the mechanisms by which these proteins are trafficked? What are the roles of these proteins in the trafficking of other polarized membrane proteins in the same cells? What are the nature of the intracellular compartments in which these proteins distribute? The Cores are an Administrative Core (Core A), a Cell Culture/Adeno and Lentivirus Preparation Core (Core B, Director M. Tse), and a Two-Photon Microscopy and Confocal FRET/FRAP Imaging Core (Core C, Director O. Kovbasnjuk). Project 1, Knockout Models of PDZ Proteins and NHE3 Regulation, PI: M. Donowitz. The hypothesis will be tested that multiple BB PDZ proteins form multiprotein NHE3 complexes which are involved dynamically in NHE3 regulation. BB PDZ domain protein knockout mice and knockdowns (shRNAi) in Caco-2 cells will examine the role of these PDZ proteins in NHE3 regulation, complex size and associated proteins. Project 2, CIC-5 in Endocytosis and NHE3/NaPi-lla Trafficking, PI: S. Guggino. A knockout mouse model of CIC-5 which mimics Dent's disease will be studied for effects on BB endocytosis and exocytosis, trafficking of megalin, NHE3, and NaPi-lla. Whether CIC-3 and/or -4 can substitute for CIC-5 and whether acidification of endosomal pH reconstitutes trafficking effects of a CIC-5 mutant which lacks Cl channel activity but has CI/H exchange activity will test which CIC-5 function is needed. Project 3, Cu Homeostasis and Cu-ATPases in Polarized Epithelia, PI: A. Hubbard. The routes of trafficking, regulation and mechanisms involved in ATPase 7A (Menkes protein) and ATPase 7B (Wilson' protein) mediated Cu efflux from polarized epithelial cells will be studied. Associated binding proteins and the role of the C-terminus in plasma membrane targeting will be examined. Increased understanding of regulation of trafficking of plasma membrane proteins in polarized epithelia will result from these studies as well as increased understanding of the diseases which occur due to abnormal function of the transport proteins studied in each Project (diarrhea, Dent's disease, Wilson's disease, Menkes disease).
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