Structural and Functional Consequences of Mutating Cysteine Residues in the Amino Terminus of Human Multidrug Resistance-associated Protein 1

Abstract

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.

The MRP family of drug efflux pumps

“…Notwithstanding evidence indicating that N-terminal deletion of the entire MSD0 does not alter several properties of the pump, it is anticipated that this domain has functions that have yet to be uncovered. Studies showing that MRP1 activity can be affected by point mutations in the extracellular portion of the N-terminus and in MSD0, and by N-terminal deletions that extend to and include the first transmembrane domain hint at this possibility, although it remains to be determined whether these perturbations influence actual drug binding and transport as opposed to preventing the assumption of correct topology in the plasma membrane (Gao et al, 1998;Yang et al, 2002;Leslie et al, 2003b).…”

Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1)

“…A truncated mutant, which lacks this domain, was functional with respect to transport activity, and similarly to the wild-type protein, localized to the basolateral membrane in polarized cells [6]. On the other hand, certain mutations in TMD0 resulted in significant conformational changes and reduced transport activity [59,180]. Based on these observations, it has been suggested that certain residues in TMD0 contribute to the maintenance of the correct structure of the protein.…”

New Use for an Old Drug: Inhibiting ABCG2 with Sorafenib

“…HEK293-transfected sublines HEK293/ Vec (18), HEK293/ABCC1 (19), and HEK293/ABCG2 (20) were obtained from Prof. Jian-ting Zhang (Indiana University Simon Cancer Center, Indianapolis, IN). The human breast cancer cell line MCF-7 and the mitoxantrone selected MDR cell line MCF-7/MX (21) were kindly provided by Dr. E. Schneider (Wadsworth Center, Albany, NY).…”