Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

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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.

Section: Discussionmentioning

confidence: 99%

Section: Effect Of Mutations Of Cys 7 and Cys 32 On Human Mrp1-mediatmentioning

confidence: 99%

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confidence: 99%

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

Yang

Chen

Zhang

2002

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“…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)(12)(13)(14). Although it has recently been argued that the amino-terminal end (33 amino acids) may form a U-shaped structure and function as a gate (15,16), the functional role of MSD0 remains to be determined.…”

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confidence: 99%

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

Yang¹,

Liu

Dong³

et al. 2007

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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.

“…The predicted extracellular location of the amino terminus of ABCC1 has been confirmed by determining glycosylation status (16,17) by epitope insertion (18,19) and by cell-free expression in microsomal membranes (20). However, this prevailing topological orientation of the amino terminus has recently been challenged by a study using a monoclonal antibody (IU2H10) against the amino terminus of human ABCC1, which suggested that part of the amino terminus was exposed intracellularly (21).…”

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confidence: 99%

The Amino Terminus of the Human Multidrug Resistance Transporter ABCC1 Has a U-shaped Folding with a Gating Function

Chen

Yang

et al. 2006

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Multidrug resistance is a serious problem in successful cancer chemotherapy. Studies using model cell lines have demonstrated that overexpression of some members of the ATP-binding cassette (ABC) transporter superfamily, such as ABCC1, causes enhanced efflux and, thus, decreased accumulation of multiple anticancer drugs, which leads to increased cell survival. Unlike most other ABC transporters, ABCC1 has an additional membrane-spanning domain (MSD0) with a putative extracellular amino terminus of 32 amino acids. However, the function of MSD0 and the role of the extracellular amino terminus are largely unknown. In this study, we examined the structural folding and the function of the amino terminus. We found that it has a U-shaped folding with the bottom of the U-structure facing cytoplasm and both ends in extracellular space. We also found that this U-shaped amino terminus probably functions as a gate to regulate the drug transport activity of human ABCC1.