Arginine-482 is not essential for transport of antibiotics, primary bile acids and unconjugated sterols by the human breast cancer resistance protein (ABCG2)

Abstract

The human BCRP (breast cancer resistance protein, also known as ABCG2) is an ABC (ATP-binding cassette) transporter that extrudes various anticancer drugs from cells, causing multidrug resistance. To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg482-->Gly) mutant BCRP (BCRP-G) in Lactococcus lactis. Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L. lactis. In agreement with observations in mammalian cells, the BCRP-R-mediated transport of the cationic substrates rhodamine 123 and tetramethylrosamine was significantly decreased compared with the activity of BCRP-G. In addition, BCRP-R showed an enhanced interaction with the anionic anticancer drug methotrexate when compared with BCRP-G, suggesting that structure/substrate specificity relationships in BCRP, as observed in eukaryotic expression systems, are maintained in prokaryotic L. lactis. Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L. lactis, for which the R482G mutation was not critical. Since Arg482 is predicted to be present in the intracellular domain of BCRP, close to transmembrane segment 3, our results point to a role of this residue in electrostatic interactions with charged substrates including rhodamine 123 and methotrexate. Since unconjugated sterols are neutral molecules and bile acids and many antibiotics are engaged in protonation/deprotonation equilibria at physiological pH, our observations may point either to a lack of interaction between Arg482 and neutral or neutralized moieties in these substrates during transport or to the interaction of these substrates with regions in BCRP not including Arg482.

ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates

“…Statistical significance relative to parental HEK293 cells: *, P < 0.05; **, P < 0.001. In contrast to these findings, Janvilisiri and his colleagues (32) have recently shown that the transport of the ABCG2 substrate Hoechst 33342 by G482-ABCG2 can be blocked by as low as 25 Amol/L methotrexate. Hence, this latter report can possibly support the suggestion that G482-ABCG2 may have the facility to accommodate and presumably transport methotrexate and other antifolates as well as their short-chain polyglutamate derivatives.…”

Down-Regulation of Organic Anion Transporter Expression in Human Hepatocytes Exposed to the Proinflammatory Cytokine Interleukin 1β

“…2B), likely indicating that at least some OATPs were targeted by IL-1␤ at mRNA, protein, and activity levels. Transporters belonging to the MRP subfamily, namely, MRP2 and MRP3, and the ABC transporter breast cancer resistance protein (ABCG2), which mediates transport of organic anions such as bilirubin diglucuronide, antibiotics, and bile acids (Janvilisri et al, 2005), were also down-regulated, although in a more moderate manner because the factors of repression ranged from 1.7-to 4.1-fold in primary hepatocytes and HepaRG cells (Table 2). In a similar manner, a 24-h exposure to IL-1␤ only moderately repressed OATP8 (SLCO1B3) mRNA levels in HepaRG cells by a 1.9-fold factor, whereas a shorter treatment time (8 h) had no effect.…”

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…Regarding potential bile salt transport by ABCG2, the experimental results are contradictory. ABCG2 expressed in Lactococcus lactis showed higher ATPase activity in the presence of certain bile salts and caused a minor bacterial resistance against bile salt toxicity ( Janvilisri, Shahi, Venter, Balakrishnan, & van Veen, 2005). In CHO cells, coexpression of rat Oatp1a1 and human ABCG2 enhanced both the uptake and the efflux of a fluorescent conjugated bile acid, cholyl-glycylamido-fluorescein, cholic acid (CA), glycoCA, tauroCA, and taurolithocholic acid-3-sulfate (Blazquez et al, 2012).…”