Ligand-mediated tertiary structure changes of reconstituted P-glycoprotein. A tryptophan fluorescence quenching analysis.
Ligand-dependent changes in accessibility of purified P-glycoprotein, functionally reconstituted in liposomes, were investigated by fluorescence measurements. Trp quenching experiments provided evidence that P-glycoprotein adopts different tertiary structures upon binding of drug substrates in the absence and presence of MgATP and its nonhydrolyzable analog, MgATPgammaS. Five anthracycline derivatives were tested as drug substrates: daunorubicin, 4'-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, and methoxy-morpholino-doxorubicin. Among them, daunorubicin and 4'-epi-doxorubicin have been shown to be rejected outside the multidrug-resistant cells, whereas the three others have been shown to accumulate in multidrug-resistant cells overexpressing P-glycoprotein and therefore retain their cytotoxic activity. A small conformational change was associated with nucleotide binding and amplified after nucleotide hydrolysis. Different conformational states were adopted by P-glycoprotein upon the addition of the anthracycline derivatives in the absence and presence of MgATP or MgATPgammaS. These conformational changes are shown to be related to the nature of the antitumor agents and more precisely to their capacity to accumulate in resistant cells. These data also suggest that the cytotoxicity of iododoxorubicin and 4-demethoxy-daunorubicin is related to the fact they are not transported by P-glycoprotein. On the contrary, methoxy-morpholino-doxorubicin cytotoxicity may be explained in terms of its rapid reincorporation into the plasma membrane after being transported by P-glycoprotein.
Sonveaux
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Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, B-1050 Brussels, Belgium.
Vigano
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C
Shapiro
A B
AB
Ling
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V
Ruysschaert
J M
JM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D020106:Acrylamide / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001386:Azides; D016466:CHO Cells; D006224:Cricetinae; D004095:Dihydropyridines; D018432:Drug Resistance, Multiple; D005453:Fluorescence; D008024:Ligands; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000903:Antibiotics, Antineoplastic; D001386:Azides; D004095:Dihydropyridines; D008024:Ligands; D009711:Nucleotides; D011510:Proteolipids; C020485:proteoliposomes; D020106:Acrylamide; C041130:azidopine; D014364:Tryptophan; D000255:Adenosine Triphosphate