Review
A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function

https://doi.org/10.1016/j.bbapap.2009.02.014Get rights and content

Abstract

The MDR1 (ABCB1) gene encodes a membrane-bound transporter that actively effluxes a wide range of compounds from cells. The overexpression of MDR1 by multidrug-resistant cancer cells is a serious impediment to chemotherapy. MDR1 is expressed in various tissues to protect them from the adverse effect of toxins. The pharmacokinetics of drugs that are also MDR1 substrates also influence disease outcome and treatment efficacy. Although MDR1 is a well-conserved gene, there is increasing evidence that its polymorphisms affect substrate specificity. Three single nucleotide polymorphisms (SNPs) occur frequently and have strong linkage, creating a common haplotype at positions 1236C>T (G412G), 2677G>T (A893S) and 3435C>T (I1145I). The frequency of the synonymous 3435C>T polymorphism has been shown to vary significantly according to ethnicity. Existing literature suggests that the haplotype plays a role in response to drugs and disease susceptibility. This review summarizes recent findings on the 3435C>T polymorphism of MDR1 and the haplotype to which it belongs. A possible molecular mechanism of action by ribosome stalling that can change protein structure and function by altering protein folding is discussed.

Introduction

Toxins in the environment are a major threat to many living organisms. Once internalized, toxic compounds must be removed for the organisms to survive. Therefore, humans have developed, inherited and perfected ways to reduce the effect of xenobiotics. At the cellular level, the cell membrane acts as a physical barrier to prevent compounds from entering the cell. However, because some compounds can diffuse through the cell membrane, alternative protective methods have evolved. One of the most important defense mechanisms is to pump xenobiotics out of the cells. Thus, drug transporters are commonly found in the cell membranes of many organisms, from bacteria to mammals, and are responsible for cell protection. However, the existence of these drug transporters often hinders the use of compounds used to treat diseases because they are substrates of these efflux pumps either by affecting the pharmacokinetics of drugs in the body or by limiting accumulation in target cells such as cancer cells.

MDR1 (P-glycoprotein, ABCB1) was the first ABC transporter identified and has become the most studied gene in the field of multidrug resistance [1]. It is a member of the ATP-binding cassette (ABC) transporter superfamily [2]. In addition to MDR1, 47 other ABC transporters have been identified or predicted in the human genome. These transporters are classified into seven groups (A to G) based on sequence similarities [2]. Among these, several (ABCA2, ABCB4, ABCG2, ABCC1–5,6,10) are also able to confer drug resistance (reviewed in [3], [4]). Sequence similarities do not predict the role of ABC transporters, suggesting that protein folding and the formation of the substrate binding pocket play important roles in their function. ABC transporters show diverse expression patterns. Human MDR1 is normally found in a tissue-specific manner. It is found in normal cells of the adrenal gland, kidney, liver, colon, jejunum, pancreas, and in capillary endothelial cells in the testis and blood brain barrier [5], [6], [7]. MDR1 is also found in the placenta and in the endometrium of pregnant women. It is expressed in a polarized manner in apical cells lining the small intestine and the colon and in the kidney proximal tubules. The expression pattern of MDR1 suggests that its major physiological role is to protect vital organs and the fetus [8] from xenobiotics.

MDR1 is highly expressed in drug-resistant cancer cells, particularly in cancer cells originating from cells that normally express MDR1 [9]. It was first detected on colchicine-resistant Chinese Hamster Ovary (CHO) cells [10], and has frequently been found in cell culture models of MDR and in clinical samples from tumors that are resistant to chemotherapy. The MDR1 gene is found on chromosome 7, at band p21–21.1 [11], and its cDNA spans about 4.5 kb, with 28 exons ranging in size from 49 to 587 bp [12]. The MDR1 gene encodes a polypeptide with 1280 amino acids which has an apparent molecular weight of 170 kDa. The protein is defined as having two halves, each containing six hydrophobic trans-membrane domains, and an ATP-binding domain (Fig. 1). The two halves are separated by a flexible linker region, and the two ATP-binding domains are structurally similar. All 12 trans-membrane domains are found in the plasma membrane. Several motifs have been identified in each of the ATP-binding domains, including the Walker-A, Walker-B, A-loop, H-loop, D-loop, Q-loop and the signature motif “LSSGQ” consensus sequences. The ATP-binding domains act as ATPases that hydrolyze ATP to ADP. In vitro studies have shown that the ATPase activity can be induced in the presence of MDR1 substrates [13], [14], [15]. MDR1 is post-translationally regulated, and contains three N-linked glycosylation sites (N91, N94 and N99) in the first extracellular loop [16]. The post-translationally processed protein is localized in the plasma membrane [17], and a glycosylation-defective mutant does not show altered drug transport [18]. Several phosphorylation sites have also been identified, but studies on mutants have shown that these sites are not responsible for localization or function in cultured cells [16], [19].

Biochemical studies suggested that substrate transport by MDR1 is coupled to ATP hydrolysis [20], [21]. There is speculation that MDR1 may work as a flippase so that substrate initially interacts with the inner leaflet of the lipid bilayer and then MDR1 flips the compound to the outer leaflet [22]. Nonetheless, the currently accepted model is the “hydrophobic vacuum pump”, in which the substrate directly interacts with the protein's drug-binding pocket and is pumped into the extracellular space, assisted by hydrolysis of ATP to ADP (reviewed in [3], [23], [24]).

The most significant feature of MDR1 function is its broad substrate specificity. Reviews have categorized MDR1 substrates based on their clinical use [25], [26], and by correlating MDR1 expression levels with drug resistance in the NCI-60 cell-line panel [27], it has been predicted that a great number of compounds are candidate MDR1 substrates [28]. These MDR1 substrates have diverse chemical structures and have a wide range of biological functions, e.g., anti-cancer drugs, anti-HIV proteases, antihistamines, calcium channel blockers, antibiotics, etc. This substrate “promiscuity”, and the absence of a crystal structure for MDR1, makes prediction of MDR1 substrates difficult and therefore attempts to predict MDR1 substrates based on chemical structures have to date been unsuccessful. The substrate binding mechanism and recognition of substrates and inhibitors by MDR1 is complex. It has been found that MDR1 contains a large drug-binding pocket that is generally assembled by several trans-membrane domains (TM5, TM6, TM11 and TM12) which determines the substrate specificity of MDR1 [29], [30], [31]. The presence of this drug-binding pocket is further supported by photoaffinity labeling experiments with MDR1 substrate analogues. However, there are mutations of amino acids in other parts of the transporter that also change substrate specificity, suggesting that drug recognition is a complex process [32], [33].

The function of MDR1 as a transporter is similar to other drug transporters. Within the long list of MDR1 substrates, it is evident that some of the substrates (drugs) are also substrates of drug metabolizing enzymes, especially CYP3A4. CYP3A4 is located at 7q22.1, very close to MDR1 on the chromosome, suggesting their inter-dependency during evolution to protect the host organism from toxins by detoxification and extrusion. Some MDR1 substrates, e.g. anti-cancer drugs, can be effluxed by other MDR transporters, suggesting redundancy, or “back up” in transporter function. For example, vincristine is a substrate of MDR1 [34] and MRP1 (ABCC1) [35] while doxorubicin is a substrate of MDR1 [36], MRP1 (ABCC1) [37] and BCRP (ABCG2) [38].

MDR1's expression pattern and function suggest that it plays a crucial role in drug absorption, disposition and elimination. Studies using transgenic knockout mice have provided important evidence that loss of MDR1 affects drug pharmacokinetics [39], [40]. Although only one MDR1 gene in humans confers drug resistance, mice have two (mdr1a, mdr1b) [41]. Transgenic mice with genetic disruption of mdr1a, mdr1b or both were generated [39], [40]. All of these animals are viable and have no significant defects. However, these mice have higher sensitivity to MDR1 substrates in vivo. Sensitivity to ivermectin, a neurotoxin, was increased 100-fold in mdr1a knockout mice [39]. The mice also showed increased sensitivity to other drugs such as dexamethasone, digoxin and cyclosporine A. The effect of drug accumulation is greater in organs expressing mdr1b in the mdr1a/1b double knockout mice [40]. This shows that both genes share common function. Importantly, loss of function of both mdr1 genes does not increase expression of other drug resistance genes in mice, indicating that change in drug accumulation is due to loss of mdr1a and mdr1b genes [40]. Clearly, this evidence strongly suggests that pharmacokinetics of many drugs is affected by mdr1a/mdr1b.

Section snippets

Characterization of MDR1 polymorphisms

According to the SNP database maintained by the National Center for Biotechnology Information (NCBI), there are more than 50 SNPs in the human MDR1 coding region. Table 1 summarizes the SNPs in the exons of MDR1 reported in both the NCBI and Ensemble databases. Studying the location of the SNPs has led to several important observations. First, SNPs with more than 1% heterozygosity were found in two-thirds of the twenty-nine total exons. SNPs are found in the MDR1 transcript from the 5′ start

Importance of understanding the role of MDR1 polymorphisms

The presence of MDR1 directly influences drug efficacy and its expression determines the degree of resistance of cancer cells to chemotherapy. Therefore, there is an urgent need to understand the factors that determine the function of MDR1. Since it is a well-conserved gene, research has been focused on the factors that affect its expression (reviewed in [47]). Mutation studies have confirmed that changes in crucial amino acid residues in the trans-membrane domains, ATP-binding domains,

Elucidating the role of the 3435C>T SNP and its haplotype

Several SNPs in the exonic region of MDR1 occur frequently, indicating their importance during the evolution of MDR1. Much interest has been focused on the polymorphism at 3435, located in the middle of exon 26. This mutation, which changes cytosine to thymine, is found frequently (Supplemental Table 1). It is a wobble mutation that translates to isoleucine, a hydrophobic amino acid residue. This isoleucine is well-conserved in different animal species, from humans to primates, mice and pigs.

3435C>T affects MDR1 function

The frequently occurring 3435C>T mutation has been extensively studied in recent years. Studies using cell lines and patient samples have suggested that this mutation leads to several changes, from mRNA level, protein expression, protein folding to substrate specificity. How does one synonymous mutation change the structure and function of MDR1 in so many ways? Recent reports have focused on the association of this synonymous SNP with changes in MDR1 transport function, and also to changes in

Possible impact of translational pauses in MDR1 haplotype

Synonymous mutation-mediated ribosome stalling may affect domains ahead of the pause sites. The ribosome is a large macromolecular complex consisting of large and small subunits. It runs along the mRNA from 5′ to 3′ catalyzing polypeptide synthesis from information recorded in mRNA. Formation of the peptide bond is found in the peptidyl transferase center (PTC) in the large subunit [105]. During translation, growing nascent polypeptides must travel through a portion of the ribosome before they

Conclusions and perspectives

In MDR1, most non-synonymous SNPs do not change protein function. There is a misconception that all synonymous SNPs are “silent” because they do not change the encoded amino acid. In fact, cumulative evidence suggests that gene functions and onset of human diseases could be affected by synonymous polymorphisms (reviewed in [118], [119]). In one MDR1 haplotype, there are two synonymous SNP sites (1236C>T and 3435C>T) that occur frequently and have a distinctive distribution pattern in human

Acknowledgements

This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute. We thank Mr. George Leiman for editorial assistance.

References (136)

  • S.V. Ambudkar et al.

    Purification and reconstitution of human P-glycoprotein

    Methods Enzymol.

    (1998)
  • F.J. Sharom et al.

    Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein

    J. Biol. Chem.

    (1993)
  • C.F. Higgins et al.

    Is the multidrug transporter a flippase?

    Trends Biochem. Sci.

    (1992)
  • Y. Raviv et al.

    Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells

    J. Biol. Chem.

    (1990)
  • G. Szakacs et al.

    Predicting drug sensitivity and resistance: profiling ABC transporter genes in cancer cells

    Cancer Cell.

    (2004)
  • X. Zhang et al.

    Functional evidence that transmembrane 12 and the loop between transmembrane 11 and 12 form part of the drug-binding domain in P-glycoprotein encoded by MDR1

    J. Biol. Chem.

    (1995)
  • C.A. Doige et al.

    ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells

    Biochim. Biophys. Acta

    (1992)
  • C.S. Morrow et al.

    Coordinated action of glutathione S-transferases (GSTs) and multidrug resistance protein 1 (MRP1) in antineoplastic drug detoxification. Mechanism of GST A1-1- and MRP1-associated resistance to chlorambucil in MCF7 breast carcinoma cells

    J. Biol. Chem.

    (1998)
  • P. Borst et al.

    Genetic dissection of the function of mammalian P-glycoproteins

    Trends Genet.

    (1997)
  • U. Potocnik et al.

    Common germline MDR1/ABCB1 functional polymorphisms and haplotypes modify susceptibility to colorectal cancers with high microsatellite instability

    Cancer Genet. Cytogenet.

    (2008)
  • T.W. Loo et al.

    Functional consequences of proline mutations in the predicted transmembrane domain of P-glycoprotein

    J. Biol. Chem.

    (1993)
  • T.W. Loo et al.

    Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains

    J. Biol. Chem.

    (2004)
  • Y. Zhou et al.

    The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2

    Arch. Biochem. Biophys.

    (1999)
  • A. Roulet et al.

    MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoprotein substrate ivermectin

    Eur. J. Pharmacol.

    (2003)
  • G.R. Lankas et al.

    P-glycoprotein deficiency in a subpopulation of CF-1 mice enhances avermectin-induced neurotoxicity

    Toxicol. Appl. Pharmacol.

    (1997)
  • D.R. Umbenhauer et al.

    Identification of a P-glycoprotein-deficient subpopulation in the CF-1 mouse strain using a restriction fragment length polymorphism

    Toxicol. Appl. Pharmacol.

    (1997)
  • N. Kioka et al.

    P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

    Biochem. Biophys. Res. Commun.

    (1989)
  • K. Saito et al.

    Detection of the four sequence variations of MDR1 gene using TaqMan MGB probe based real-time PCR and haplotype analysis in healthy Japanese subjects

    Clin. Biochem.

    (2003)
  • V. Eswaran et al.

    Genomics refutes an exclusively African origin of humans

    J. Hum. Evol.

    (2005)
  • N.N. Salama et al.

    MDR1 haplotypes significantly minimize intracellular uptake and transcellular P-gp substrate transport in recombinant LLC-PK1 cells

    J. Pharm. Sci.

    (2006)
  • C.J. Tsai et al.

    Synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima

    J. Mol. Biol.

    (2008)
  • H. Grosjean et al.

    Preferential codon usage in prokaryotic genes: the optimal codon–anticodon interaction energy and the selective codon usage in efficiently expressed genes

    Gene

    (1982)
  • S. Varenne et al.

    The maximum rate of gene expression is dependent on the downstream context of unfavourable codons

    Biochimie

    (1989)
  • V. Ramachandiran et al.

    Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro

    FEBS Lett.

    (2002)
  • T. Ikemura

    Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes

    J. Mol. Biol.

    (1981)
  • R. Allikmets et al.

    Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database

    Hum. Mol. Genet.

    (1996)
  • S.V. Ambudkar et al.

    P-glycoprotein: from genomics to mechanism

    Oncogene

    (2003)
  • G. Szakacs et al.

    Targeting multidrug resistance in cancer

    Nat. Rev., Drug. Discov.

    (2006)
  • F. Thiebaut et al.

    Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues

    Proc. Natl. Acad. Sci. U. S. A.

    (1987)
  • F. Thiebaut et al.

    Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein

    J. Histochem. Cytochem.

    (1989)
  • A.T. Fojo et al.

    Expression of a multidrug-resistance gene in human tumors and tissues

    Proc. Natl. Acad. Sci. U. S. A.

    (1987)
  • N. Kartner et al.

    Daunorubicin-resistant Chinese hamster ovary cells expressing multidrug resistance and a cell-surface P-glycoprotein

    Cancer. Res.

    (1983)
  • A. Fojo et al.

    Localization of multidrug resistance-associated DNA sequences to human chromosome 7

    Somat. Cell. Mol. Genet.

    (1986)
  • J.J. Gribar et al.

    Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system

    J. Membr. Biol.

    (2000)
  • S.V. Ambudkar et al.

    Biochemical, cellular, and pharmacological aspects of the multidrug transporter

    Annu. Rev. Pharmacol. Toxicol.

    (1999)
  • T. Sakaeda et al.

    MDR1 genotype-related pharmacokinetics and pharmacodynamics

    Biol. Pharm. Bull.

    (2002)
  • C. Marzolini et al.

    Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance

    Clin. Pharmacol. Ther.

    (2004)
  • R.H. Shoemaker

    The NCI60 human tumour cell line anticancer drug screen

    Nat. Rev. Cancer.

    (2006)
  • S. Kajiji et al.

    Functional analysis of P-glycoprotein mutants identifies predicted transmembrane domain 11 as a putative drug binding site

    Biochemistry

    (1993)
  • T.W. Loo et al.

    Mutations to amino acids located in predicted transmembrane segment 6 (TM6) modulate the activity and substrate specificity of human P-glycoprotein

    Biochemistry

    (1994)
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