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.
Showing the most recent 10 out of 20 publications
