Regulation of Function by Dimerization through the Amino-terminal Membrane-spanning Domain of Human ABCC1/MRP1

Youyun Yang,Yang Liu,Zizheng Dong,Junkang Xu,Hui Peng,Zhaoqian Liu,Jian-Ting Zhang

doi:10.1074/jbc.m700152200

Abstract

Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCB1/P-glycoprotein/MDR1 and ABCC1/MRP1 causes multidrug resistance in cancer chemotherapy. It has been thought that half-ABC transporters with one nucleotide-binding domain and one membrane-spanning domain (MSD) likely work as dimers, whereas full-length transporters with two nucleotide-binding domains and two or three MSDs function as monomers. In this study, we examined the oligomeric status of the human full-length ABC transporter ABCC1/MRP1 using several biochemical approaches. We found 1) that it is a homodimer, 2) that the dimerization domain is located in the amino-terminal MSD0L0 (where L0 is loop 0) region, and 3) that MSD0L0 has a dominant-negative function when coexpressed with wild-type ABCC1/MRP1. These findings suggest that ABCC1/MRP1 may exist and function as a dimer and that MSD0L0 likely plays some structural and regulatory functions. It is also tempting to propose that the MSD0L0-mediated dimerization may be targeted for therapeutic development to sensitize ABCC1/MRP1-mediated drug resistance in cancer chemotherapy.

Highlights

Unlike most of other human ABC transporters such as ABCB1/P-glycoprotein/MDR1, which contain a core structure of MSD1-NBD1-MSD2-NBD2 (Fig. 1), ABCC1, as well as ABCC2, ABCC3, ABCC6, and ABCC8 –10, contains an additional MSD (MSD0) at the amino terminus that consists of five predicted transmembrane segments with a putative extracellular amino-terminal end (11–14)
We examined the oligomeric status of human ABCC1 using multiple approaches, including perfluorooctanoic acid (PFO)-PAGE, nondenaturing PAGE, gel filtration chromatography, sucrose density gradient sedimentation, chemical cross-linking, and co-immunoprecipitation
We demonstrate that human ABCC1 is a homodimer and that MSD0L0 is essential and sufficient for homodimerization of human ABCC1

Summary

Introduction

(ABCB1/MDR1) and MRP1 (multidrug resistance-associated protein 1; ABCC1) causes multidrug resistance. Fractions (0.5 ml) were collected, followed by trichloroacetic acid precipitation, separation by SDS-PAGE, transfer to PVDF membranes, and detection of ABCC1 by Western blot analysis using monoclonal antibody MRPr1 as a probe.

Results

Conclusion