Abstract
Multidrug resistance protein 1 (MRP1) (gene symbol ABCC1) is an ATP-binding cassette (ABC) transporter which effluxes xeno- and endobiotic organic anions including estradiol glucuronide and the pro-inflammatory leukotriene C4. MRP1 also confers multidrug resistance by reducing intracellular drug accumulation through active efflux. MRP1 has three membrane spanning domains (MSD), and two nucleotide binding domains (NBD). MSD1 and MSD2 are linked to NBD1 and NBD2 by connecting regions (CR) 1 and CR2, respectively. Here we targeted four residues in CR1 (Ser612, Arg615, His622, Glu624) for alanine substitution and unexpectedly, found that cellular levels of three mutants (S612A, R615A, E624A) in transfected HEK cells were substantially lower than wild-type MRP1. Whereas CR1-H622A properly trafficked to the plasma membrane and exhibited organic anion transport activity comparable to wild-type MRP1, the poorly expressing R615A and E624A (and to a lesser extent S612A) mutant proteins were retained intracellularly. Analyses of cryogenic electron microscopic and atomic homology models of MRP1 indicated that Arg615 and Glu624 might participate in bonding interactions with nearby residues to stabilize expression of the transporter. However, this was not supported by double exchange mutations E624K/K406E, R615D/D430R and R615F/F619R which failed to improve MRP1 levels. Nevertheless, these experiments revealed that the highly conserved CR1-Phe619 and distal Lys406 in the first cytoplasmic loop of MSD1 are also essential for expression of MRP1 protein. This study is the first to demonstrate that CR1 contains several highly conserved residues critical for plasma membrane expression of MRP1 but thus far, currently available structures and models do not provide any insights into the underlying mechanism(s). Additional structures with rigorous biochemical validation data are needed to fully understand the bonding interactions critical to stable expression of this clinically important ABC transporter.
Highlights
The ATP-binding cassette (ABC) multidrug resistance protein 1 (MRP1) is expressed at varying levels in most tissues where it effluxes a wide variety of physiologic and xenobiotic organic anions [1,2,3]
It confers resistance to these drugs by reducing their cellular accumulation [7]. It plays a lesser role than the ABC drug transporting P-glycoprotein (ABCB1) and ABCG2, MRP1-mediated drug efflux is believed to be clinically relevant in some multidrug resistant tumors and in the tissue distribution of various therapeutic agents [8, 9]
To determine which CR1 residues to target for alanine substitution, multiple sequence alignments of MRP1 CR1 with the comparable region of bovine Mrp1, the eight other transporting ABCC subfamily members [58, 59], as well as the three non-transporting ABCC subfamily members were carried out using Clustal Omega [60] (Fig 2B–2D)
Summary
The ATP-binding cassette (ABC) multidrug resistance protein 1 (MRP1) (encoded by ABCC1) is expressed at varying levels in most tissues where it effluxes a wide variety of physiologic and xenobiotic organic anions [1,2,3]. It confers resistance to these drugs by reducing their cellular accumulation [7] It plays a lesser role than the ABC drug transporting P-glycoprotein (ABCB1) and ABCG2, MRP1-mediated drug efflux is believed to be clinically relevant in some multidrug resistant tumors and in the tissue distribution of various therapeutic agents [8, 9]. Distinct from P-glycoprotein and ABCG2, MRP1-mediated export of natural product drugs and some other xenobiotics and organic anions requires the presence of the physiological tripeptide antioxidant GSH (γ-Glu-Cys-Gly) [10,11,12,13,14,15]. GSH and its oxidized dimer glutathione disulfide (GSSG) are themselves transported by MRP1 [16,17,18,19,20,21], suggesting potential roles for MRP1 in a wide variety of GSH/GSSG dependent cellular processes including enzyme function, signal transduction, apoptosis, ferroptosis, protein biosynthesis and assembly as well as neutralization of nitric oxide and reactive oxygen species [22,23,24,25,26,27,28,29,30]
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