Two members of the mouse mdr gene family confer multidrug resistance with overlapping but distinct drug specificities.

We report the cloning and functional analysis of a complete clone for the third member of the mouse mdr gene family, mdr3. Nucleotide and predicted amino acid sequence analyses showed that the three mouse mdr genes encode highly homologous membrane glycoproteins, which share the same length (1,276 residues), the same predicted functional domains, and overall structural arrangement. Regions of divergence among the three proteins are concentrated in discrete segments of the predicted polypeptides. Sequence comparison indicated that the three mouse mdr genes were created from a common ancestor by two independent gene duplication events, the most recent one producing mdr1 and mdr3. When transfected and overexpressed in otherwise drug-sensitive cells, the mdr3 gene, like mdr1 and unlike mdr2, conferred multidrug resistance to these cells. In independently derived transfected cell clones expressing similar amounts of either MDR1 or MDR3 protein, the drug resistance profile conferred by mdr3 was distinct from that conferred by mdr1. Cells transfected with and expressing MDR1 showed a marked 7- to 10-fold preferential resistance to colchicine and Adriamycin compared with cells expressing equivalent amounts of MDR3. Conversely, cells transfected with and expressing MDR3 showed a two- to threefold preferential resistance to actinomycin D over their cellular counterpart expressing MDR1. These results suggest that MDR1 and MDR3 are membrane-associated efflux pumps which, in multidrug-resistant cells and perhaps normal tissues, have overlapping but distinct substrate specificities.     

Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdr1 and mdr2 genes.

A full-length cDNA clone for the mouse mdr1 gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdr1 which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdr1 and analyzed these chimeras for their capacity to transfer drug resistance. The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdr1. Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdr1 could also confer drug resistance. However, the replacement of either the amino- or carboxy-terminus transmembrane (TM) domain regions of mdr1 by the homologous segments of mdr2 resulted in inactive chimeras. The replacement of as few as two TM domains from either the amino (TM5-6) or the carboxy (TM7-8) half of mdr1 by the homologous mdr2 regions was sufficient to destroy the activity of mdr1. These results suggest that the functional differences detected between mdr1 and mdr2 in our transfection assay reside within the predicted TM domains.     

Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins

The complete nucleotide and primary structure (1276 amino acids) of a full length mdr cDNA capable of conferring a complete multidrug-resistant phenotype is presented. The deduced amino acid sequence suggests that mdr is a membrane glycoprotein which includes six pairs of transmembrane domains and a cluster of potentially N-linked glycosylation sites near the amino terminus. A striking feature of the protein is an internal duplication that includes approximately 500 amino acids. Each duplicated segment includes a consensus ATP-binding site. Amino acid homology is observed between the mdr gene and a series of bacterial transport genes. This strong homology suggests that a highly conserved functional unit involved in membrane transport is present in the mdr polypeptide. We propose that an energy-dependent transport mechanism is responsible for the multidrug-resistant phenotype.     

A staphylococcal multidrug resistance gene product is a member of a new protein family.

The complete nucleotide sequence (321 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates. The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The smr gene was subcloned and expressed in S. aureus and E. coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes: phosphonium derivatives and quarternary amines. Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations. The deduced polypeptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr). Four transmembrane segments are predicted for smr gene product. Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment. This Glu 13 is conserved in all members of the family of small membrane proteins. We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation. This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylic residues in the transmembrane segments of Smr.     

Isolation and characterization of Drosophila multidrug resistance gene homologs.

Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp. The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances. This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene. These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins. Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism. This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.     

Sequence of mdr3 cDNA encoding a human P-glycoprotein

We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.     

Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily.

STE6, the yeast a-factor transporter, is a member of the ATP binding cassette protein superfamily, which also includes the mammalian multidrug resistance protein and the cystic fibrosis gene product. These proteins contain two homologous halves, each with six membrane spanning segments and a predicted ATP nucleotide binding domain. To assess the importance of the two halves of STE6, and to examine the functional significance of residues conserved among members of the ATP binding cassette superfamily, we introduced mutations into the nucleotide binding domains of STE6. Our analysis demonstrates that both halves of STE6 are critical for function and that some, but not all, mutations analogous to those known to result in cystic fibrosis impair STE6 activity. To examine further the functional contribution of each half of the STE6 protein, we severed the STE6 coding sequence and expressed the two halves of the transporter as separate polypeptides. Whereas 'half-molecules' are unable to provide transport function individually, co-expression of both half-molecules in the same cell leads to functional reconstitution of STE6-mediated a-factor transport.     

Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.     

The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene

We present the complete nucleotide sequence and the deduced amino acid sequence of the H-2Dp class I gene. This gene, which was cloned from a B10.P genomic DNA library, encodes and intact, functional H-2Dp molecule. Comparative analysis of the Dp sequence with other class I sequences reveals both similarities and differences. This analysis also shows that these genes exhibit D region-specific, locus-specific, as well as allele-specific sequences. The H-2Dp nucleotide sequence is greater than 90% homologous to the H-2Ld and H-2Db genes and only approximately 85% homologous to the H-2Dd gene. The K region and Qa region genes are less homologous. The 3' noncoding sequences appear to be region-specific. All of the previously described D region genes, Db, Ld, and Dd, possess the B2-SINE Alu-like repetitive sequence, as does Dp. Thus, this B2 repeat is a region-specific marker present in all D region genes studied so far. The additional polyadenylation site found in the H-2Dp gene starting at nucleotide 4671, which is homologous to non-D region sequences, as well as unique protein Dp coding sequences, make this gene an interesting model for studying the evolution of polymorphism and structure/function relationships in the class I gene family.     

Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells

Resistance of tumor cells to multiple cytotoxic drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human cells is determined by the mdr1 gene, encoding a high molecular weight membrane glycoprotein (P-glycoprotein). Complete primary structure of human P-glycoprotein has been determined from the cDNA sequence. The protein, 1280 amino acids long, consists of two homologous parts of approximately equal length. Each half of the protein includes a hydrophobic region with six predicted transmembrane segments and a hydrophilic region. The hydrophilic regions share homology with peripheral membrane components of bacterial active transport systems and include potential nucleotide-binding sites. These results are consistent with a function for P-glycoprotein as an energy-dependent efflux pump responsible for decreased drug accumulation in multidrug-resistant cells.     

Molecular cloning of a novel human leukemia-associated gene. Evidence of conservation in animal species

We have recently described an 18-kilodalton polypeptide (p18) that is present in much greater abundance in acute leukemic blast cells (myeloid and lymphoid) than in resting or proliferating nonleukemic lymphoid cells or chronic lymphoid and myeloid leukemic cells. In this report we describe the cloning of two different sized full-length cDNAs that code for p18. The two cDNAs differ in their 3'-noncoding regions as a result of alternative polyadenylation. Analysis of the complete nucleotide sequence and the corresponding amino acid sequence did not reveal significant homology to any previously described sequences. We show evidence that this gene is highly conserved in several animal species and low stringency hybridization studies suggest that the p18 gene may be a member of a family of partially homologous genes in the human genome.     

Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA

Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.