Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway

Kazuhiro Katayama, Khyati Kapoor, Shinobu Ohnuma, Atish Patel, William Swaim, Indu S. Ambudkar, Suresh V. Ambudkar

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

Original languageEnglish
Pages (from-to)2361-2370
Number of pages10
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1853
Issue number10
DOIs
Publication statusPublished - 2015 Oct 1
Externally publishedYes

Keywords

  • Degradation
  • Endosome
  • Half-life
  • Lysosome
  • P-glycoprotein
  • Proteasome

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

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