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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

Gál, Zita and Hegedüs, Csilla and Szakács, Gergely and Váradi, András and Sarkadi, Balázs and Laczka, Csilla (2015) 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. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1848 (2). pp. 477-487. ISSN 0005-2736

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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.

Item Type: Article
Uncontrolled Keywords: sterol sensing; regulatory mechanism; Protein Transport; protein motif; protein modification; protein interaction; PROTEIN FUNCTION; protein expression; Protein Binding; nonhuman; MOLECULAR RECOGNITION; MOLECULAR MECHANICS; mammal cell; insect cell; human cell; human; enzyme activity; controlled study; Consensus Sequence; ARTICLE; animal cell; amino acid sequence; tyrosine derivative; breast cancer resistance protein; bile acid; adenosine triphosphatase; multidrug resistance; Cholesterol recognition amino acid consensus; Cholesterol; Bile acids; ABCG2
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 07 Jul 2015 14:12
Last Modified: 07 Jul 2015 14:12
URI: http://real.mtak.hu/id/eprint/25138

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