Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

This chapter deals with the interactions of two medically important multidrug ABC transporters (MDR-ABC), ABCB1 and ABCG2, with lipid molecules. Both ABCB1 and ABCG2 are capable of transporting a wide range of hydrophobic drugs and xenobiotics and are involved in cancer chemotherapy resistance. Therefore, the exploration of their mechanism of action has major therapeutic consequences. As discussed here in detail, both ABCB1 and ABCG2 are significantly affected by various lipid compounds especially those residing in their close proximity in the plasma membrane. ABCB1 is capable of transporting lipids and lipid derivatives, and thus may alter the general membrane composition by "flopping" membrane lipid constituents, while there is no such information regarding ABCG2. Still, both ABCB1 and ABCG2 show complex interactions with a variety of lipid molecules, and the transporters are significantly modulated by cholesterol and cholesterol derivatives at the posttranslational level. In this chapter, we explore the molecular details of the direct transporter-lipid interactions, the potential role of lipid-sensor domains within the proteins, as well as the application of experimental site-directed mutagenesis, detailed structural studies, and in silico modeling for examining these interactions. We also discuss the regulation of ABCB1 and ABCG2 expression at the transcriptional level, occurring through nuclear receptors involved in lipid sensing. The better understanding of lipid interactions with these medically important MDR-ABC transporters may significantly improve further drug development and clinical treatment options

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

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

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INST: L_08

Regulation of the Function of the Human ABCG2 Multidrug Transporter by Cholesterol and Bile Acids: Effects of Mutations in Potential Substrate- And Steroid-Binding Sites.

ABCG2 is a plasma membrane glycoprotein that actively extrudes xenobiotics and endobiotics from the cells and causes multidrug resistance in cancer. In the liver, ABCG2 is expressed in the canalicular membrane of hepatocytes and excretes its substrates into the bile. ABCG2 is known to require high membrane cholesterol content for maximal activity and, by examining purified ABCG2 reconstituted in proteoliposomes, we have recently shown that cholesterol is an essential activator, while bile acids significantly modify the activity of this protein. In the present work, by using isolated insect cell membrane preparations expressing the human ABCG2 and its mutant variants, we have analyzed whether certain regions in this protein are involved in the sterol recognition. We found that replacing ABCG2-R482 with large amino acids does not affect cholesterol-dependence, while changes to small amino acids cause altered cholesterol sensitivity. When leucines in the potential steroid-binding element (SBE, aa 555-558) of ABCG2 were replaced by alanines, cholesterol-dependence of ABCG2 activity was strongly reduced, although the L558A mutant, when purified and reconstituted, still required cholesterol for full activity. Regarding the effects of bile acids in isolated membranes, we found that these compounds decreased ABCG2-ATPase in the absence of drug substrates, while did not significantly affect substrate-stimulated ATPase activity. The above ABCG2 mutant variants also altered bile acid sensitivity, although cholic acid and glycocholate are not transported by the protein. We suggest that the aforementioned two regions in ABCG2 are important for sterol sensing and may represent potential targets for pharmacological modulation of ABCG2 function