The goal of this 5-year proposal is to elucidate the structural, cellular, and environmental factors that may modulate the activity of the intestinal dipeptide transporter, PepT1. A 12- transmembrane domain, proton-coupled transporter protein, PepT1 is known to play an important role in the transport of nutritional di- and tripeptides and such therapeutically important, structurally-related drugs as beta-lactam antibiotics and angiotensin converting enzyme inhibitors. We will test the hypothesis that specific amino acid residues in hPepT1 determine substrate and H+ translocation, trafficking of the transporter protein, and its modulation. There are three specific aims in this research: (1) To establish the structure-function relationship of PepT1; (2) To elucidate the sorting pathway of PepT1 in polarized epithelial cells; and (3) To investigate the factors that modulate the function of PepT1 in Caco-2 cells. In preliminary studies, we have identified three amino acids (Y167, E595, and W294) that may be key to transporter function, demonstrated localization of PepT1 in both apical plasma membrane and lysosomes in several cell types, and demonstrated regulation of PepT1 activity possibly by protein kinase C (PKC). Experimentally, to address Aim number 1 on structure-function of PepT1, we will use a synergistic combination of computer modeling, site-directed mutagenesis and 3H-Gly-Sar uptake studies in HEK293 cells transiently transfected with the cDNA of PepT1. To determine whether a given mutation affects substrate or H+ recognition, translocation and release, we will use a panel of PepT1 substrates to assess uptake and the two-microelectrode voltage-clamp technique in Xenopus oocytes to monitor transient charge movement. To address Aim number 2 on sorting of the wild- type and mutated hPepT1, we will use subcellular fractionation, immunostaining, scanning alanine mutagenesis of candidate sorting motifs, and biotinylation of metabolically pulse-labeled PepT1. To address Aim number 3 on modulation of PepT1 activity, we will use activators and inhibitors of protein kinase C, perform site- directed mutagenesis of putative phosphorylation sites, and evaluate the role of microtubules and microfilaments in PepT1 trafficking. The significance of this research is that it will (a) improve the mechanistic understanding of peptide translocation through PepT1, (b) facilitate the design of drugs targeted for PepT1, and (c) serve as the molecular basis for the search for human polymorphism of PepT1.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059297-02
Application #
6363306
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Okita, Richard T
Project Start
2000-03-01
Project End
2004-02-28
Budget Start
2001-03-01
Budget End
2002-02-28
Support Year
2
Fiscal Year
2001
Total Cost
$325,999
Indirect Cost
Name
University of Southern California
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Uchiyama, Tomomi; Fujita, Takuya; Gukasyan, Hovhannes J et al. (2008) Functional characterization and cloning of amino acid transporter B(0,+) (ATB(0,+)) in primary cultured rat pneumocytes. J Cell Physiol 214:645-54
Links, Jennifer L S; Kulkarni, Ashutosh A; Davies, Daryl L et al. (2007) Cysteine scanning of transmembrane domain three of the human dipeptide transporter: implications for substrate transport. J Drug Target 15:218-25
Price, Eric A; Sutch, Brian T; Cai, Qi et al. (2007) Computation of nitroxide-nitroxide distances in spin-labeled DNA duplexes. Biopolymers 87:40-50
Kulkarni, Ashutosh A; Davies, Daryl L; Links, Jennifer S et al. (2007) A charge pair interaction between Arg282 in transmembrane segment 7 and Asp341 in transmembrane segment 8 of hPepT1. Pharm Res 24:66-72
da Costa, Silvia R; Wu, Kaijin; Veigh, Michelle Mac et al. (2006) Male NOD mouse external lacrimal glands exhibit profound changes in the exocytotic pathway early in postnatal development. Exp Eye Res 82:33-45
Wu, Kaijin; Jerdeva, Galina V; da Costa, Silvia R et al. (2006) Molecular mechanisms of lacrimal acinar secretory vesicle exocytosis. Exp Eye Res 83:84-96
Rentsendorj, A; Xie, J; MacVeigh, M et al. (2006) Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer. Gene Ther 13:821-36
Jerdeva, Galina V; Wu, Kaijin; Yarber, Francie A et al. (2005) Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells. J Cell Sci 118:4797-812
Wang, Y; Xie, J; Yarber, F A et al. (2004) Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland. Gene Ther 11:970-81
Chu, Chun; Okamoto, Curtis T; Hamm-Alvarez, Sarah F et al. (2004) Stable transfection of MDCK cells with epitope-tagged human PepT1. Pharm Res 21:1970-3

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