In this PPG Renewal Application submitted after 2 years of funding, 6 NIH-funded PI's propose interactive research focusing on the regulation of outward trafficking particularly to the apical domain of specific transport proteins in polarized epithelial cells from the small intestine, liver, lung and kidney. Four 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), the intracellular Cl channel CIC-5 (Project 2), ATPase7B (Wilson protein) (Project 3), and CFTR (newly added Project 4). The common questions being addressed are: How is the function of each protein regulated by plasma membrane trafficking or how does the protein regulate plasma trafficking of other transport proteins? What are the nature of the intracellular compartments in which these proteins occur? What proteins do these transporters interact with as part of regulated trafficking? The Cores are an Administrative Core (Core A, Director, M. Donowitz), Cell Culture/Adeno- and Lentivirus Preparation Core (Core B, Director, M. Tse), and Advanced Imaging (Two-Photon Microscopy, Confocal Microscopy, FRET/FRAP) 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 protein knockout mice and knockdowns of individual NHERF proteins will be studied in Caco-2 cells to examine the role of these PDZ proteins in NHE3 regulation. Project 2, CIC-5 in Exocytosis and Endocytosis and in NHES/Megalin Trafficking (PI, S. Guggino). A knockout mouse model of ClC-5 which mimics Dent disease and was shown to have defects in exocytosis as well as endocytosis will be studied for mechanisms of effects on BB trafficking of NHE3, megalin, and NaPi2A. Mechanistic studies of how ClC-5 is involved in regulated exocytosis will examine the role of Rab 11b. Project 3, Cu Homeostasis and ATPase7B in Polarized Epithelia (PI, A. Hubbard). The routes of trafficking, regulation and mechanisms involved in ATPase7B (Wilson protein) mediated Cu efflux from polarized epithelial will be studied. Associated binding proteins and the role of the N- and C-terminus in plasma membrane targeting will be examined. Project 4, Outward trafficking of CFTR (PI, William Guggino). The processing and outward trafficking of CFTR, its B band, and the mutant (508 will be studied for how the PDZ domain containing proteins CAL and the NHERFs coordinate setting plasma membrane and organellar distribution of CFTR under basal and cAMP-stimulated conditions.
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 trafficking and function ofthe transport proteins studied in each Project (NHE3: diarrhea. Congenital Na diarrhea;CIC-5: Dent disease;ATPase7B: Wilson disease;CFTR:CF).
|Gupta, Arnab; Schell, Michael J; Bhattacharjee, Ashima et al. (2016) Myosin Vb mediates Cu+ export in polarized hepatocytes. J Cell Sci 129:1179-89|
|In, Julie; Foulke-Abel, Jennifer; Zachos, Nicholas C et al. (2016) Enterohemorrhagic Escherichia coli reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell Mol Gastroenterol Hepatol 2:48-62.e3|
|Zachos, Nicholas C; Kovbasnjuk, Olga; Foulke-Abel, Jennifer et al. (2016) Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology. J Biol Chem 291:3759-66|
|Foulke-Abel, Jennifer; In, Julie; Yin, Jianyi et al. (2016) Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology. Gastroenterology 150:638-649.e8|
|Cebotaru, Liudmila; Liu, Qiangni; Yanda, Murali K et al. (2016) Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease. Kidney Int 90:90-9|
|Braiterman, Lelita T; Gupta, Arnab; Chaerkady, Raghothama et al. (2015) Communication between the N and C termini is required for copper-stimulated Ser/Thr phosphorylation of Cu(I)-ATPase (ATP7B). J Biol Chem 290:8803-19|
|Janecke, Andreas R; Heinz-Erian, Peter; Yin, Jianyi et al. (2015) Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea. Hum Mol Genet 24:6614-23|
|Chen, Tiane; Kocinsky, Hetal S; Cha, Boyoung et al. (2015) Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site. J Biol Chem 290:1952-65|
|Zhu, Xinjun Cindy; Sarker, Rafiquel; Horton, John R et al. (2015) Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity. Am J Physiol Cell Physiol 308:C758-66|
|Yang, Jianbo; Sarker, Rafiquel; Singh, Varsha et al. (2015) The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity. Biochem J 470:77-90|
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