Cytoplasmic retraction of the amino terminus of human multidrug resistance protein 1.
Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) transport superfamily which also includes human multidrug resistance 1 (MDR1) gene product P-glycoprotein (Pgp). Overexpression of MRP1 or Pgp causes multidrug resistance in cancer cells. Different from Pgp, MRP1 contains an extra membrane-spanning domain (MSD1) with a putative extracellular amino terminus in addition to the core structure of two MSDs and two NBDs (nucleotide-binding domains). The structural and functional significance of the additional MSD1 in MRP1 remains elusive. In this study, we generated an IgG1 subclass monoclonal antibody, IU2H10, specific to the amino terminus of human MRP1 and mapped its epitope to 10 amino acids (S8ADGSDPLWD17). It can be used for Western blot, immunoprecipitation, and indirect immunofluorescence studies of human MRP1. However, surprisingly we found that IU2H10 cannot react with MRP1 unless cells are permeabilized. Furthermore, the IU2H10 epitope is exposed extracellularly when the carboxyl-terminal core domain of human MRP1 is deleted. Examination of the amino-terminal sequence of human MRP1 suggests that it consist of mainly coiled structures. These observations provide evidence for a model that is different from the prevailing extracellular location of the amino terminus of human MRP1. It is possible that part of the amino terminus of human MRP1, following exposure to the lumen of the endoplasmic reticulum, is retracted to the cytoplasm.
Department of Pharmacology and Toxicology, Walther Oncology Center/Walther Cancer Institute and Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Qun
Q
Chen
Youyun
Y
Yang
Yang
Y
Liu
Baoguang
B
Han
Jian-Ting
JT
Zhang
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D000939:Epitopes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C421109:multidrug resistance-associated protein 1