title: Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface.
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'-(beta,gamma-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.
authors.affiliation: National Medical Center, Institute of Haematology and Immunology, Membrane Research Group and Hungarian Academy of Sciences, 1113 Budapest, Hungary.
authors.forename: Csilla
authors.initials: C
authors.lastname: Ozvegy-Laczka
authors.forename: György
authors.initials: G
authors.lastname: Várady
authors.forename: Gabriella
authors.initials: G
authors.lastname: Köblös
authors.forename: Olga
authors.initials: O
authors.lastname: Ujhelly
authors.forename: Judit
authors.initials: J
authors.lastname: Cervenak
authors.forename: John D
authors.initials: JD
authors.lastname: Schuetz
authors.forename: Brian P
authors.initials: BP
authors.lastname: Sorrentino
authors.forename: Gerrit-Jan
authors.initials: GJ
authors.lastname: Koomen
authors.forename: András
authors.initials: A
authors.lastname: Váradi
authors.forename: Katalin
authors.initials: K
authors.lastname: Német
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001562:Benzimidazoles / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007313:Insecta; D008024:Ligands; D008942:Mitoxantrone / Q000494:pharmacology; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000502:physiology*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013997:Time Factors; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D001562:Benzimidazoles; D008024:Ligands; D009363:Neoplasm Proteins; D000266:Adenylyl Imidodiphosphate; D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008942:Mitoxantrone; D000251:Adenosine Triphosphatases; C017807:bisbenzimide ethoxide trihydrochloride