Structural and functional consequences of mutating cysteine residues in the amino terminus of human multidrug resistance-associated protein 1.
Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette membrane transport superfamily and is responsible for multidrug resistance in cancer cells. Currently, there are nine known human MRPs. Distinct from many other members of the ATP-binding cassette superfamily, human MRP1 and four other MRPs have an additional membrane-spanning domain (MSD) with a putative extracellular amino terminus. The functional significance of this additional MSD (MSD1) is currently unknown. To understand the role of MSD1 in human MRP1 structure and function, we studied the amino-terminal 33 amino acids. We found that the amino terminus of human MRP1 has two cysteine residues (Cys(7) and Cys(32)) that are conserved among the five human MRPs that have MSD1. Mutation analyses of the two cysteines in human MRP1 revealed that the Cys(7) residue is critical for the MRP1-mediated drug resistance and leukotriene C(4) transport activity. On the other hand, mutation of Cys(32) reduced only moderately the MRP1 function. The effect of Cys(7) mutation on MRP1 activity appears to be due to the 5-7-fold decrease in the maximal transport rate V(max). We also found that mutation of Cys(7) changed the amino-terminal conformation of MRP1. This conformational change is likely responsible for the decrease in V(max) of LTC(4) transport mediated by the mutant MRP1. Based on these studies, we conclude that the amino terminus of human MRP1 is important and that the Cys(7) residue plays a critical role in maintaining the proper structure and function of human MRP1.
Yang
Youyun
Y
Department of Pharmacology and Toxicology, Walther Oncology Center/Walther Cancer Institute and the Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis 46202, USA.
Chen
Qun
Q
Zhang
Jian-Ting
JT
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D014750:Vincristine / Q000494:pharmacology
D000255:Adenosine Triphosphate; D000409:Alanine; D000970:Antineoplastic Agents; D003078:Colchicine; D003545:Cysteine; D004317:Doxorubicin; D005047:Etoposide; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014357:Trypsin; D014750:Vincristine; C421109:multidrug resistance-associated protein 1