Oligomerization of the human ABC transporter ABCG2: evaluation of the native protein and chimeric dimers.
Human ABCG2, a member of the ATP binding cassette (ABC) transporter superfamily, is overexpressed in numerous multidrug-resistant cells in culture. Localized to the plasma membrane, ABCG2 contains six transmembrane segments and one nucleotide binding domain (NBD) and is thought to function as a dimer or higher order oligomer. Chimeric fusion proteins containing two ABCG2 proteins joined either with or without a flexible linker peptide were expressed at the plasma membrane and maintained drug transport activity. Expression of an ABCG2 variant mutated in a conserved residue in the Walker B motif of the NBD (D210N) resulted in a non-functional protein expressed at the cell surface. Expression of an ABCG2 chimeric dimer containing the D210N mutation in the first ABCG2 resulted in a dominant-negative phenotype, as the protein was expressed at the surface but was not functional. Using a bifunctional photoaffinity nucleotide analogue and a non-membrane-permeable cysteine-specific chemical cross-linking agent, a dimer is the predominant form of oligomerized ABCG2 under our assay conditions. Furthermore, these experiments demonstrated that the dimer interface includes, but may not be limited to, interactions between residues in each monomeric NBD and separate disulfide interactions between the cysteines in the third extracellular loop of each monomer. By changing all three extracellular cysteines to alanine, we showed that although extracellular disulfide bonds may exist between monomers, they are not essential for ABCG2 localization, transport activity, or prazosin-stimulated ATPase activity. Together, these data suggest that ABCG2 functions as a dimer, but do not exclude functional higher order oligomers.
Bhatia
Aarti
A
Department of Chemistry and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907-2084, USA.
Schäfer
Hans-Jochen
HJ
Hrycyna
Christine A
CA
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D019281:Dimerization; D004351:Drug Resistance / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014162:Transfection
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases