Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface

C. Özvegy-Laczka, György Várady, Gabriella Köblös, Olga Ujhelly, Judit Cervenak, John D. Schuetz, Brian P. Sorrentino, Gerrit Jan Koomen, A. Váradi, K. Német, B. Sarkadi

Research output: Contribution to journalArticle

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Abstract

The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein.

Original languageEnglish
Pages (from-to)4219-4227
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number6
DOIs
Publication statusPublished - Feb 11 2005

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Adenosine Triphosphate
Monoclonal Antibodies
Vanadates
Antibodies
Adenylyl Imidodiphosphate
Adenosine Diphosphate
Membranes
Proteins
Multiple Drug Resistance
Adenosine
Labeling
Adenosine Triphosphatases
Conformations
Tumors
Membrane Proteins
Nucleotides
Modulation
ATP Binding Cassette Transporter, Sub-Family G, Member 2
Ligands
Substrates

ASJC Scopus subject areas

  • Biochemistry

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Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface. / Özvegy-Laczka, C.; Várady, György; Köblös, Gabriella; Ujhelly, Olga; Cervenak, Judit; Schuetz, John D.; Sorrentino, Brian P.; Koomen, Gerrit Jan; Váradi, A.; Német, K.; Sarkadi, B.

In: Journal of Biological Chemistry, Vol. 280, No. 6, 11.02.2005, p. 4219-4227.

Research output: Contribution to journalArticle

Özvegy-Laczka, C. ; Várady, György ; Köblös, Gabriella ; Ujhelly, Olga ; Cervenak, Judit ; Schuetz, John D. ; Sorrentino, Brian P. ; Koomen, Gerrit Jan ; Váradi, A. ; Német, K. ; Sarkadi, B. / Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 6. pp. 4219-4227.
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AU - Várady, György

AU - Köblös, Gabriella

AU - Ujhelly, Olga

AU - Cervenak, Judit

AU - Schuetz, John D.

AU - Sorrentino, Brian P.

AU - Koomen, Gerrit Jan

AU - Váradi, A.

AU - Német, K.

AU - Sarkadi, B.

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