Cancer Letters

Cancer Letters

Volume 162, Issue 2, 26 January 2001, Pages 181-191
Cancer Letters

Analysis of the structure and expression pattern of MRP7 (ABCC10), a new member of the MRP subfamily

https://doi.org/10.1016/S0304-3835(00)00646-7Get rights and content

Abstract

The MRP subfamily of ABC transporters currently consists of at least six members, several of which have been demonstrated to transport amphipathic anions and to confer in vitro resistance to chemotherapeutic agents. In searching the data bases we identified the product of a cDNA sequencing project that bears significant similarity to MRP subfamily transporters. In this report the predicted coding sequence, protein product and expression pattern of this cDNA, termed MRP7, are analyzed. The MRP7 cDNA sequence encodes a 1492 amino acid ABC transporter whose structural architecture resembles that of MRP1, MRP2, MRP3, and MRP6, in that its transmembrane helices are arranged in three membrane spanning domains. However, in contrast to the latter transporters, a conserved N-linked glycosylation site is not found at the N-terminus of MRP7. Comparisons of the MRP7 amino acid sequence indicated that while it is most closely related to other MRP subfamily members, its degree of relatedness is the lowest of any of the known MRP-related transporters. The integrity of the predicted MRP7 coding sequence was confirmed by the synthesis of a ~ 158 kDa protein in reticulocyte lysates programmed with the MRP7 cDNA. While MRP7 transcript was detected in a variety of tissues by RT/PCR, it was not readily detectable by RNA blot analysis, suggesting that it is expressed at low levels in these tissues. Fluorescence in situ hybridization indicated that MRP7 maps to chromosome 6p12-21, in proximity to several genes associated with glutathione conjugation and synthesis. On the basis of these findings and evolutionary cluster analysis, we conclude that MRP7 is a member of the MRP subfamily of amphipathic anion transporters.

Introduction

Cellular resistance to anticancer agents is a significant obstacle to the chemotherapeutic treatment of disseminated human cancers. Plasma membrane transporters that function to reduce intracellular drug concentrations represent one class of cellular resistance mechanisms. Pgp, a 170 kD protein that confers resistance to a wide range of natural product anticancer agents, has served as a paradigm for this class of resistance mechanisms [1]. More recently amphipathic anion transporters such as MRP1 and MRP2 (or cMOAT) [2], [3], [4], [5] have been implicated in drug resistance. These two proteins function as primary active transporters of glutathione and glucuronate conjugates as well as several other amphipathic anions [6], [7], [8], [9], [10], [11], [12], [13]. The drug resistance activities of MRP1 and MRP2 overlap with that of Pgp, but are distinguished by their inability to confer resistance to Taxol and by their capacity to confer resistance to some agents that are not part of the Pgp profile [14], [15], [16], [17], [18], [19]. In contrast to Pgp, MRP1 and MRP2 confer resistance to methotrexate and camptothecins, and MRP2 has also been reported to confer resistance to cisplatin [17], [20], [21]. The mechanism by which MRP1 and MRP2 efflux natural product agents differs from that of Pgp in that they are thought to cotransport drugs with glutathione [9], [22], [23], whereas cotransported molecules are not required for drug transport by Pgp.

Analyses of EST sequences and other partial sequences indicate that MRP1 and MRP2 are members of a large subfamily that consists of at least five additional human members [24], [25], [26]. Our laboratory and others have reported the complete coding sequences and expression patterns of four of these five human transporters [27], [28], [29], [30], [31], [32], [33] and two have been described in the rat [34]. Characterizations of some of the more recently described MRP family members indicate that while they also have the capacity to transport amphipathic anions, their substrate selectivities [35], [36], [37], [38], [39] and drug resistance profiles [40], [41], [42], [43], [44], [45] are distinct from those of MRP1 and MRP2.

Of the five ESTs and partial sequences reported to date, the full length coding sequence of only one remains to be described (EST182763) [24]. In searching the data bases we identified a nucleotide sequence that is the product of a cDNA sequencing project and which bears significant similarity to MRP subfamily transporters. In this report the predicted protein encoded by this cDNA, termed MRP7, is analyzed and the expression pattern of its transcript is examined. In so doing we determined that MRP7 is a human MRP subfamily member that corresponds to EST182763. MRP7 transcript is expressed at very low levels in a variety of human tissues examined, encodes a protein of 158 kDa in reticulocyte lysates, and shares the protein topology of MRP1, MRP2, MRP3 and MRP6. Characterization of MRP7 helps to further define the MRP subfamily of transporters.

Section snippets

Computer methods

The Blast program was used to identify data base sequences that are related to EST182763. Protein sequence analyses, including comparisons, alignments and cluster analysies, were performed using the Wisconsin Package version 9.1 (Genetics Computer Group, WI). For the GAP program, the Blosum 62 scoring matrix was used with a gap creation penalty of 8 and a gap extension penalty of 2. These improved parameters differ slightly from those used in our previous analyses of the MRP subfamily [28], [29]

Predicted structure of MRP7

Data base analysis revealed a nucleotide sequence (GenBank accession BAA92227) that shares a high degree of similarity with MRP subfamily transporters. Nucleotides 3131-3633 of this data base submission were identical to EST 182763, the MRP7-related EST for which a complete coding sequence has yet to be described [24]. Analysis of revealed an open reading frame of 4479 nucleotides that was preceded by in frame stop codons at nucleotides 11 and 50. The putative initiation codon was preceded by

Discussion

The analysis we present here indicates that MRP7 is a seventh member of the MRP subfamily. Within this subfamily, the MRP7 domain structure is similar to that of MRP1, MRP2, MRP3, and MRP6, in that it possesses an N-terminal membrane spanning domain [29]. However, pairwise comparisons of amino acid sequences indicate that among the subfamily members MRP7 has the least structural resemblance to MRP1. Nevertheless, its inclusion in this subfamily as determined by cluster analysis and amino acid

Acknowledgements

The work was supported by NIH grant CA73728 to GDK, NIH grant CA 06927 to Fox Chase cancer Center and by an appropriation from the Commonwealth of Pennsylvania.

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