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.

Public Health Relevance

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).

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Program Projects (P01)
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Special Emphasis Panel (ZDK1-GRB-8 (M2))
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Hamilton, Frank A
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Johns Hopkins University
Internal Medicine/Medicine
Schools of Medicine
United States
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Cha, Boyoung; Chen, Tiane; Sarker, Rafiquel et al. (2014) Lysophosphatidic acid stimulation of NHE3 exocytosis in polarized epithelial cells occurs with release from NHERF2 via ERK-PLC-PKC? signaling. Am J Physiol Cell Physiol 307:C55-65
Braiterman, Lelita T; Murthy, Amrutha; Jayakanthan, Samuel et al. (2014) Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B. Proc Natl Acad Sci U S A 111:E1364-73
Nyasae, Lydia K; Schell, Michael J; Hubbard, Ann L (2014) Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes. Traffic 15:1344-65
Yang, Jianbo; Singh, Varsha; Chen, Tian-E et al. (2014) NHERF2/NHERF3 protein heterodimerization and macrocomplex formation are required for the inhibition of NHE3 activity by carbachol. J Biol Chem 289:20039-53
Singh, Varsha; Yang, Jianbo; Chen, Tiane-e et al. (2014) Translating molecular physiology of intestinal transport into pharmacologic treatment of diarrhea: stimulation of Na+ absorption. Clin Gastroenterol Hepatol 12:27-31
Li, Xuhang; Donowitz, Mark (2014) Fractionation of subcellular membrane vesicles of epithelial and non-epithelial cells by OptiPrep™ density gradient ultracentrifugation. Methods Mol Biol 1174:85-99
Singh, Varsha; Lin, Rong; Yang, Jianbo et al. (2014) AKT and GSK-3 are necessary for direct ezrin binding to NHE3 as part of a C-terminal stimulatory complex: role of a novel Ser-rich NHE3 C-terminal motif in NHE3 activity and trafficking. J Biol Chem 289:5449-61
Chen, Tiane; Hubbard, Ann; Murtazina, Rakhilya et al. (2014) Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells. J Cell Sci 127:3535-45
Foulke-Abel, Jennifer; In, Julie; Kovbasnjuk, Olga et al. (2014) Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract. Exp Biol Med (Maywood) 239:1124-34
Cheng, Jie; Guggino, William (2013) Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6. PLoS One 8:e68001

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