P-Glycoprotein conformational changes detected by antibody competition.
Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.
Nagy
H
H
Department of Biophysics and Cell Biology, University of Debrecen, Hungary.
Goda
K
K
Arceci
R
R
Cianfriglia
M
M
Mechetner
E
E
Szabó
G
G
D016475:3T3 Cells; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000906:Antibodies / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001667:Binding, Competitive; D002121:Calcium Channel Blockers / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D009543:Nifedipine / Q000494:pharmacology; D011136:Polysorbates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014634:Valinomycin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000900:Anti-Bacterial Agents; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000972:Antineoplastic Agents, Phytogenic; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D011136:Polysorbates; D014634:Valinomycin; D014747:Vinblastine; D016572:Cyclosporine; D014700:Verapamil; D009543:Nifedipine