Effects of ATP depletion and phosphate analogues on P-glycoprotein conformation in live cells

K. Goda, Henrietta Nagy, Eugene Mechetner, Maurizio Cianfriglia, Gábor Szabó

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

P-glycoprotein (Pgp), a membrane pump often responsible for the multidrug resistance of cancer cells, undergoes conformational changes in the presence of substrates/modulators, or upon ATP depletion, reflected by its enhanced reactivity with the UIC2 monoclonal antibody. When the UIC2-shift was elicited by certain modulators (e.g. cyclosporin A or vinblastine, but not with verapamil or Tween 80), the subsequent binding of other monoclonal anti-Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E. & Szabó Jr, G. (2001) Eur. J. Biochem. 268, 2416-2420]. To further study the relationship between UIC2-shift and the suppression of MM12.10 binding, we compared, on live cells, how ATP depletion and treatment of cells with phosphate analogues (sodium orthovanadate, beryllium fluoride and fluoro-aluminate) that trap nucleotides at the catalytic site, affect the two phenomena. Similarly to modulators or ATP depleting agents, all the phosphate analogues increased daunorubicin accumulation in Pgp-expressing cells. Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10, in accordance with the enhanced binding of the first mAb. Vanadate and beryllium fluoride, but not fluoro-aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10. Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in response to direct modulation of the ATP-binding site, confirming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2-related phenomena. These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state.

Original languageEnglish
Pages (from-to)2672-2677
Number of pages6
JournalEuropean Journal of Biochemistry
Volume269
Issue number11
DOIs
Publication statusPublished - 2002

Fingerprint

P-Glycoprotein
Conformations
Adenosine Triphosphate
Phosphates
Modulators
Vanadates
Cyclosporine
Epitopes
Cells
Daunorubicin
Vinblastine
Polysorbates
Multiple Drug Resistance
Verapamil
Labeling
Catalytic Domain
Nucleotides
Sodium
Binding Sites
Monoclonal Antibodies

Keywords

  • Conformation
  • MM12.10
  • Multidrug resistance
  • P-glycoprotein
  • UIC2

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of ATP depletion and phosphate analogues on P-glycoprotein conformation in live cells. / Goda, K.; Nagy, Henrietta; Mechetner, Eugene; Cianfriglia, Maurizio; Szabó, Gábor.

In: European Journal of Biochemistry, Vol. 269, No. 11, 2002, p. 2672-2677.

Research output: Contribution to journalArticle

Goda, K. ; Nagy, Henrietta ; Mechetner, Eugene ; Cianfriglia, Maurizio ; Szabó, Gábor. / Effects of ATP depletion and phosphate analogues on P-glycoprotein conformation in live cells. In: European Journal of Biochemistry. 2002 ; Vol. 269, No. 11. pp. 2672-2677.
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AB - P-glycoprotein (Pgp), a membrane pump often responsible for the multidrug resistance of cancer cells, undergoes conformational changes in the presence of substrates/modulators, or upon ATP depletion, reflected by its enhanced reactivity with the UIC2 monoclonal antibody. When the UIC2-shift was elicited by certain modulators (e.g. cyclosporin A or vinblastine, but not with verapamil or Tween 80), the subsequent binding of other monoclonal anti-Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E. & Szabó Jr, G. (2001) Eur. J. Biochem. 268, 2416-2420]. To further study the relationship between UIC2-shift and the suppression of MM12.10 binding, we compared, on live cells, how ATP depletion and treatment of cells with phosphate analogues (sodium orthovanadate, beryllium fluoride and fluoro-aluminate) that trap nucleotides at the catalytic site, affect the two phenomena. Similarly to modulators or ATP depleting agents, all the phosphate analogues increased daunorubicin accumulation in Pgp-expressing cells. Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10, in accordance with the enhanced binding of the first mAb. Vanadate and beryllium fluoride, but not fluoro-aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10. Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in response to direct modulation of the ATP-binding site, confirming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2-related phenomena. These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state.

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