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 , 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.

Role of the Breast Cancer Resistance Protein (BCRP/ABCG2) in Drug Transport—an Update

“…2). Docking calculations indicate that Arg 482 directly interacts with mitoxantrone and Hoechst33342, but not with prazosin and SN-38 (86). This is consistent with previous findings that resistance to mitoxantrone was increased, but resistance to SN-38 or efflux of prazosin was not affected, by mutations of Arg 482 (16,87).…”

“…We found that Ala substitution of Pro 485 in TM3 significantly reduced efflux of BODIPY-prazosin by 70%, but had no effect on efflux of mitoxantrone and Hoechst 33342 (94 (96,98). However, another study showed that the activity loss caused by mutations of Lys 86 was possibly due to altered subcellular localization and cell surface targeting of BCRP (97). More information about mutations and their effects on function and expression of BCRP can be found in an open access database (http://abcmutations.hegelab.org/).…”

Structure and Function of the Human Breast Cancer Resistance Protein (BCRP/ABCG2)

“…[87] demonstrated that the TM5-loop-TM6 fragment may play a critical role in BCRP oligomerization. A monoclonal antibody 5D3 that recognizes a conformational sensitive epitope in this extracellular loop has been highly useful for biochemical characterization of BCRP in terms of interactions of the transporter with substrates or inhibitors [63, 102, 104] or conformational changes induced by point mutations [105]. …”

“…First of all, docking calculations of various BCRP substrates to the closed apo form did suggest the existence of multiple substrate binding sites in the central cavity which is primarily formed by TM α-helices [105]. Second, Arg 482 has been extensively analyzed by site-directed mutagenesis and found to be crucial for substrate specificity and transport activity [106–109].…”

Adjudin disrupts spermatogenesis by targeting drug transporters

“…Docking calculations done on BCRP and mitoxantrone (an antineoplastic agent for treating metastatic breast cancer, acute myeloid leukemia and non-Hodgkin’s lymphoma) indicate that Arg482 might be directly involved in drug interaction 21 . Another study also indicated that His457 and Arg465 might be directly involved in substrate binding 22 . These residues were thus used for setting up docking grid.…”