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Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.

ABSTRACT: The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in V(max)/K(m) for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His(457) and Arg(465) might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.

SUBMITTER: Cai X

PROVIDER: S-EPMC2879943 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.

Cai Xiaokun X Bikadi Zsolt Z Ni Zhanglin Z Lee Eun-Woo EW Wang Honggang H Rosenberg Mark F MF Mao Qingcheng Q

The Journal of pharmacology and experimental therapeutics 20100304 3

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. ...[more]

PMID: 20203106

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Project description:The ability of hydrophilic residues to shift the transverse position of transmembrane (TM) helices within bilayers was studied in model membrane vesicles. Transverse shifts were detected by fluorescence measurements of the membrane depth of a Trp residue at the center of a hydrophobic sequence. They were also estimated from the effective length of the TM-spanning sequence, derived from the stability of the TM configuration under conditions of negative hydrophobic mismatch. Hydrophilic residues (at the fifth position in a 21-residue hydrophobic sequence composed of alternating Leu and Ala residues and flanked on both ends by two Lys) induced transverse shifts that moved the hydrophilic residue closer to the membrane surface. At pH 7, the dependence of the extent of shift upon the identity of the hydrophilic residue increased in the order: L < G approximately = Y approximately = T < R approximately = H < S < P < K < E approximately = Q < N < D. By varying pH, shifts with ionizable residues fully charged or uncharged were measured, and the extent of shift increased in the order: L < G approximately = Y approximately = H(o) approximately = T < E(o) approximately = R < S < P < K+ < Q approximately = D(o) approximately = H+ < N approximately = E- < D-. The dependence of transverse shifts upon hydrophilic residue identity was consistent with the hypothesis that shift magnitude is largely controlled by the combination of side chain hydrophilicity, ionization state, and ability to position polar groups near the bilayer surface (snorkeling). Additional experiments showed that shift was also modulated by the position of the hydrophilic residue in the sequence and the hydrophobicity of the sequence moved out of the bilayer core upon shifting. Combined, these studies show that the insertion boundaries of TM helices are very sensitive to sequence, and can be altered even by weakly hydrophilic residues. Thus, many TM helices may have the capacity to exist in more than one transverse position. Knowledge of the magnitudes of transverse shifts induced by different hydrophilic residues should be useful for design of mutagenesis studies measuring the effect of transverse TM helix position upon function.

Dataset Information

Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.

Publications

Role of basic residues within or near the predicted transmembrane helix 2 of the human breast cancer resistance protein in drug transport.

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