Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein.
An hPepT1-GFP fusion construct was made to study drug inhibition of dipeptide uptake and apical, basolateral, or subcellular hPepT1 localization. The hPepT1 stop codon was mutated by polymerase chain reaction and was subsequently cloned into the pEGFP-N1 vector. The hPepT1-GFP fusion construct was then transfected into Caco-2 and HeLa cells, and drug inhibition was studied by inhibiting 3H-Gly-Sar uptake. Western blot analysis was used to determine hPepT1-GFP expression levels and confocal microscopy was used to examine the localization. Both anti-hPepT1 antibody and anti-GFP antibody recognized a 120-kd hPepT1-GFP fusion protein in the transfected cells. The 3H-Gly-Sar uptake in transfected HeLa cells was enhanced more than 20 times compared with the control. Valacyclovir (5 mmol/L) was able to completely inhibit 3H-Gly-Sar uptake in these transfected cells. Confocal microscopy showed that the hPepT1-GFP mainly localized in the Caco-2 cell apical membrane, but was present throughout the entire HeLa cell membranes. The hPepT1-GFP fusion protein was not found in either early endosome or lysosome of Caco-2 cells under normal conditions; however, it was detected in some subsets of lysosomes and early endosomes in phorbol 12-myristate 13-acetate (PMA)-treated Caco-2 cells.
Sun
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Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.
Landowski
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CP
Chu
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Wallsten
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Komorowski
T E
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Fleisher
D
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Amidon
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GL
D000212:Acyclovir / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002506:Cephalexin / Q000494:pharmacology; D003864:Depression, Chemical; D004151:Dipeptides / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D018613:Microscopy, Confocal; D000074011:Peptide Transporter 1; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D027981:Symporters; D014162:Transfection; D000077483:Valacyclovir; D014633:Valine / Q000031:analogs & derivatives* / Q000494:pharmacology
D002352:Carrier Proteins; D004151:Dipeptides; D008164:Luminescent Proteins; D000074011:Peptide Transporter 1; D011993:Recombinant Fusion Proteins; C082186:SLC15A1 protein, human; D027981:Symporters; D049452:Green Fluorescent Proteins; C004194:glycylsarcosine; D014633:Valine; D000077483:Valacyclovir; D002506:Cephalexin; D000212:Acyclovir