Expression and Function of Human MRP1 (ABCC1) Is Dependent on Amino Acids in Cytoplasmic Loop 5 and Its Interface with Nucleotide Binding Domain 2

Surtaj H Iram,Susan P.C Cole

doi:10.1074/jbc.m110.166959

Surtaj H Iram, Susan P.C Cole

Open Access

https://doi.org/10.1074/jbc.m110.166959

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Journal: Journal of Biological Chemistry	Publication Date: Mar 1, 2011
Citations: 30	License type: cc-by

Affiliation: Queen's University

Abstract

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.

Highlights

Rived from both endogenous metabolites and exogenous xenobiotics, many of which are conjugated to glutathione (e.g. the cysteinyl leukotriene (LTC4)) or glucuronide (e.g. estradiol glucuronide (E217␤G)) (4 – 6)
Sequence Alignment and Location of CL5 Residues Targeted for Mutagenesis—Our recent homology models of the core structure of Multidrug resistance protein 1 (MRP1) developed based on the crystal structure of nucleotide-bound Sav1866 from Staphylococcus aureus indicate that the ␣-helices containing TM9 and TM10 extend into the cytoplasm to become what we have designated as CL5 (Fig. 1, A and B) [11]
Mutational analyses of selected amino acids in CL5 of MSD1 and NBD2 were conducted to investigate the potential role of CL5 itself as well as the interface between these two domains in determining both the expression and activity of MRP1

Summary

Introduction

Rived from both endogenous metabolites and exogenous xenobiotics, many of which are conjugated to glutathione (e.g. the cysteinyl leukotriene (LTC4)) or glucuronide (e.g. estradiol glucuronide (E217␤G)) (4 – 6). Substitutions of several charged amino acids in CL7 substantially reduce the plasma membrane expression of MRP1 (i.e. Arg1166 and Asp1183), cause substrate-selective changes in its transport activity (i.e. Asp1179 and Glu1144), or do both (i.e. Lys1141) [22, 23].

Results

Conclusion