Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein

Duxin Sun, Christopher P. Landowski, Xiaoyan Chu, Richard Wallsten, Thomas E. Komorowski, David Fleisher, Gordon L. Amidon

Research output: Contribution to journalArticleScientificpeer-review

11 Citations (Scopus)

Abstract

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 120kd 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.
Original languageEnglish
Pages (from-to)9-17
JournalAAPS PharmSci
Volume3
Issue number1
DOIs
Publication statusPublished - 1 Dec 2001
MoE publication typeNot Eligible

Fingerprint

Caco-2 Cells
HeLa Cells
valacyclovir
Endosomes
Lysosomes
Confocal Microscopy
Pharmaceutical Preparations
Anti-Idiotypic Antibodies
Proteins
Cell Membrane
Terminator Codon
Dipeptides
Acetates
Western Blotting
Polymerase Chain Reaction
glycylsarcosine

Keywords

  • Apical membrane
  • hPepT1
  • Localization
  • Lysosome
  • Valacyclovir

Cite this

Sun, D., Landowski, C. P., Chu, X., Wallsten, R., Komorowski, T. E., Fleisher, D., & Amidon, G. L. (2001). Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein. AAPS PharmSci, 3(1), 9-17. https://doi.org/10.1208/ps030102
Sun, Duxin ; Landowski, Christopher P. ; Chu, Xiaoyan ; Wallsten, Richard ; Komorowski, Thomas E. ; Fleisher, David ; Amidon, Gordon L. / Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein. In: AAPS PharmSci. 2001 ; Vol. 3, No. 1. pp. 9-17.
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Sun, D, Landowski, CP, Chu, X, Wallsten, R, Komorowski, TE, Fleisher, D & Amidon, GL 2001, 'Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein', AAPS PharmSci, vol. 3, no. 1, pp. 9-17. https://doi.org/10.1208/ps030102

Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein. / Sun, Duxin; Landowski, Christopher P.; Chu, Xiaoyan; Wallsten, Richard; Komorowski, Thomas E.; Fleisher, David; Amidon, Gordon L.

In: AAPS PharmSci, Vol. 3, No. 1, 01.12.2001, p. 9-17.

Research output: Contribution to journalArticleScientificpeer-review

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AU - Fleisher, David

AU - Amidon, Gordon L.

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