title: Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.
abstract: Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., H-1117 Budapest, Hungary.
authors.forename: Zita
authors.initials: Z
authors.lastname: Gál
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., H-1117 Budapest, Hungary; Molecular Biophysics Research Group, Hungarian Academy of Sciences and Semmelweis University, Diószegi út 64., H-1113 Budapest, Hungary.
authors.forename: Csilla
authors.initials: C
authors.lastname: Hegedüs
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., H-1117 Budapest, Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Szakács
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., H-1117 Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Váradi
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., H-1117 Budapest, Hungary; Molecular Biophysics Research Group, Hungarian Academy of Sciences and Semmelweis University, Diószegi út 64., H-1113 Budapest, Hungary.
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudósok körútja 2., H-1117 Budapest, Hungary. Electronic address: laczka.csilla@ttk.mta.hu.
authors.forename: Csilla
authors.initials: C
authors.lastname: Özvegy-Laczka
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry*; D017124:Conserved Sequence; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012694:Serine / Q000737:chemistry / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001647:Bile Acids and Salts; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D002784:Cholesterol
keywords: ABCG2; Bile acids; Cholesterol; Cholesterol recognition amino acid consensus; Multidrug resistance