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 (Cys7 and Cys32) that are conserved among the five human MRPs that have MSD1. Mutation analyses of the two cysteines in human MRP1 revealed that the Cys7 residue is critical for the MRP1-mediated drug resistance and leukotriene C4 transport activity. On the other hand, mutation of Cys32 reduced only moderately the MRP1 function. The effect of Cys7 mutation on MRP1 activity appears to be due to the 5-7-fold decrease in the maximal transport rate Vmax. We also found that mutation of Cys7 changed the amino-terminal conformation of MRP1. This conformational change is likely responsible for the decrease in Vmax of LTC4 transport mediated by the mutant MRP1. Based on these studies, we conclude that the amino terminus of human MRP1 is important and that the Cys7 residue plays a critical role in maintaining the proper structure and function of human MRP1.