CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS

CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS CHAPTER 3 – HUMAN AND DROSOPHILA ABC PROTEINS

47 CHAPTER HUMAN AND DROSOPHILA ABC PROTEINS MICHAEL DEAN, ANDREY RZHETSKY AND RANDO ALLIKMETS INTRODUCTION From the sequencing of the human (Lander et al., 2001; Venter et al., 2001), Drosophila (Myers et al., 2000), and Caenorhabditis elegans genomes (Consortium, 1998) the full complement of ABC genes in each of these species has been characterized Figure 3.1 is an attempt to A1 A2 A4 B4 B11 C2 C6 C7/CFTR G1 G2 G4 G5 G8 Figure 3.1 Anatomy of human ABC proteins A diagram of the human body is shown with the ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 portray the major locations of some of the protein products of these genes in the human body The eukaryotic ABC genes are organized either as full transporters containing two sets of transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs), or as half transporters containing one TMD and one NBD (Hyde et al., 1990) Half transporters must form location of selected ABC transporters ABC genes with a clearly defined tissue expression and/or disease association are shown ABCA2 and ABCG4 are highly expressed in the brain, ABCA4 is exclusively expressed in the retina and mutations cause several retinal disorders CFTR is expressed in the lung and pancreas and CF patients display pathologies in these organs as well as in the intestine and the vas deferens (not shown) ABCC6 is expressed in the kidney (and also liver, not shown), but leads to pathologies in the skin, eyes, and arteries (not shown) ABCG5 and ABCG8 are expressed in the liver and intestine, and mutations in these genes lead to aberrant sterol transport in these organs ABCB4, ABCB11, ABCC2 and ABCG1 are expressed in the liver and play a role in the transport of bile components ABCA1 is expressed in peripheral cells and liver and regulates cholesterol transport ABCG2 is expressed in the placenta (not shown) and the intestine and probably serves to transport xenobiotics and toxic cell metabolites (This figure was prepared by Barking Dog Art, Gloucestershire.) Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 48 ABC PROTEINS: FROM BACTERIA TO MAN either homodimers or heterodimers in order to produce a functional transporter ABC genes are abundant in all vertebrate and invertebrate eukaryotic genomes, indicating that most of these genes have existed since the beginning of eukaryotic evolution The genes can be divided into subfamilies based on similarity in gene structure (half versus full transporters), order of the domains and sequence homology in the NBDs and TMDs There are seven mammalian ABC gene subfamilies, five of which are also found in the Saccharomyces cerevisiae genome PHYLOGENETIC ANALYSIS OF HUMAN ABC GENES The identification of the complete set of 48 human ABC genes (Table 3.1) (Dean et al., 2001) has allowed a comprehensive phylogenetic analysis of the superfamily Figure 3.2 shows a neighbor-joining tree displaying the relationships of all human ABC genes The nomenclature of ABC transporters is in TABLE 3.1 LIST OF HUMAN ABC GENES, CHROMOSOMAL LOCATION AND FUNCTION Family Symbol Alias Location Expression Function ABCA ABCA1 ABCA2 ABCA3 ABCA4 ABCA5 ABCA6 ABCA7 ABCA8 ABCA9 ABCA10 ABCA12 ABCA13 ABC1 ABC2 ABC3, ABCC ABCR 9q31.1 9q34.4 16p13.3 1p21.3 17q24.3 17q24.3 19p13.3 17q24.3 17q24.3 17q24.3 2q34 7p12.3 Ubiquitous Brain Lung Rod photoreceptors Muscle, heart, testes Liver Spleen, thymus Ovary Heart Muscle, heart Stomach Low in all tissues Cholesterol efflux onto HDL Drug resistance Surfactant production N-retinylidiene-PE efflux ABCB ABCB1 ABCB2 ABCB3 ABCB4 ABCB5 ABCB6 ABCB7 ABCB8 ABCB9 ABCB10 ABCB11 PGY1, MDR TAP1 TAP2 PGY3 Adrenal, kidney, brain All cells All cells Liver Ubiquitous Mitochondria Mitochondria Mitochondria Heart, brain Mitochondria Liver Multidrug resistance Peptide transport Peptide transport Phosphatidylcholine transport MTABC2 SPGP 7q21.12 6p21 6p21 7q21.12 7p21.1 2q35 Xq21-q22 7q36.1 12q24.31 1q42.13 2q24.3 ABCC1 ABCC2 ABCC3 ABCC4 ABCC5 ABCC6 CFTR ABCC8 ABCC9 MRP1 MRP2 MRP3 MRP4 MRP5 MRP6 ABCC7 SUR SUR2 16p13.12 10q24.2 17q21.33 13q32.1 3q27.1 16p13.12 7q31.31 11p15.1 12p12.1 Lung, testes, PBMC Liver Lung, intestine, liver Prostate Ubiquitous Kidney, liver Exocrine tissues Pancreas Heart, muscle Drug resistance Organic anion efflux Drug resistance Nucleoside transport Nucleoside transport ABCC MTABC3 ABC7 MABC1 Iron transport Fe/S cluster transport Bile salt transport Chloride ion channel Sulfonylurea receptor (continued) HUMAN AND DROSOPHILA ABC PROTEINS TABLE 3.1 (continued) Family Symbol Alias Location Expression Function ABCC10 ABCC11 ABCC12 MRP7 MRP8 MRP9 6p21.1 16q12.1 16q12.1 Low in all tissues Low in all tissues Low in all tissues ABCD ABCD1 ABCD2 ABCD3 ABCD4 ALD ALDL1, ALDR PXMP1,PMP70 PMP69, P70R Xq28 12q11 1p22.1 14q24.3 Peroxisomes Peroxisomes Peroxisomes Peroxisomes VLCFA transport regulation ABCE ABCE1 OABP, RNS4I 4q31.31 Ovary, testes, spleen Oligoadenylate-binding protein ABCF ABCF1 ABCF2 ABCF3 ABC50 6p21.1 7q36.1 3q27.1 Ubiquitous Ubiquitous Ubiquitous ABCG ABCG1 ABCG2 ABCG4 ABCG5 ABCG8 ABC8, White ABCP, MXR, BCRP White2 White3 21q22.3 4q22 11q23 2p21 2p21 Ubiquitous Placenta, intestine Liver Liver, intestine Liver, intestine Cholesterol transport? Toxin efflux, drug resistance Sterol transport Sterol transport HDL, high density lipoprotein; VLCFA, very long chain fatty acid excellent agreement with the phylogenetic groups obtained In particular, all major ABC transporter families are represented in the human tree by stable clusters with high bootstrap values This analysis provides evidence for frequent domain duplication of ATP-binding domains in ABC transporters In nearly all cases, both ATPbinding domains encoded within a gene are more closely related to each other than to ATPbinding domains from ABC transporter genes of other subfamilies This is unlikely to represent a concerted evolution of domains within the same gene, as the two domains within each gene are usually substantially diverged A far more likely scenario suggests several independent duplication events rather than a single ancestral duplication DROSOPHILA ABC GENES Analysis of the Drosophila genome sequence identified 56 ABC genes (Dean et al., 2001) with at least one representative of each of the known mammalian subfamilies (Table 3.2) To confirm the subfamily groupings the ATPbinding domain amino acid sequences were used to perform phylogenetic analyses (full transporters are represented with two ATPbinding domains each; Figure 3.3) Genes from the same subfamily cluster together and confirm the initial assignments made by inspection Both the human and Drosophila ABC genes are largely dispersed in the genome (Figures 3.4 and 3.5) In the human genome there are five clusters of two genes and one cluster of five genes For Drosophila ABC genes there are four clusters of two genes and one cluster of four genes One of these Drosophila clusters (on chromosome 2L, band 37B9) is composed of an ABCB and an ABCC gene, indicating that this is a chance grouping of genes The remaining clusters are composed of genes from the same subfamily and arranged in a head-to-tail fashion consistent with gene duplication The one exception is the human ABCG5 and ABCG8 genes, which are arranged head-to-head (Berge et al., 2000) Since the clusters themselves are dispersed and involve different subfamilies they presumably represent independent gene duplication events There are 15 ABCG genes in the Drosophila genome, making this the most abundant ABC subfamily This is in sharp contrast to the only and known ABCG genes in the human and mouse genomes, respectively The Drosophila ABCG genes are highly dispersed in the genome with only two pairs of linked genes In addition, they are quite divergent phylogenetically, suggesting that there were many 49 50 ABC PROTEINS: FROM BACTERIA TO MAN 100 29 11 ABC B1_2 ABC B4_2 ABC B5_2 55 29 65 76 61 96 ABCB8 99 ABCB3 ABCB9 ABCB6_1 ABCB7 ABC06_2 74 54 96 13 96 32 57 30 14 94 94 25 29 99 89 15 83 94 17 28 73 92 100 ABCB I ABCC7_2 ABCC10_2 ABCC11_2 ABCC12_2 ABCC5_2 100 ABCC8_2 ABCC9_2 ABCC4_2 ABCC2_2 ABCC1_2 ABCC3_2 ABCC10_1 ABCC4_1 ABCC7_1 ABCC6_1 100 ABCC8_1 ABCC9_1 ABCC1_1 ABCC2_1 ABCC3_1 ABCC5_1 ABCC11_1 ABCC12_1 ABCD4 ABCD3 92 86 II ABC B11_2 ABCB5_1 ABCB11_1 ABCB1_1 ABCB4_1 ABCD1 ABCD2 100 ABCF1_1 I ABCF3_1 ABCF2_1 ABCF3_1 ABCF1_2 II ABCF2_2 ABCE1 ABCG2 ABCG8 ABCG5 ABCG ABCG1 ABCG4 ABCA8_1 ABCA9_1 ABCC II ABCC I ABCD 100 100 69 63 49 99 70 66 86 80 100 67 62 96 100 53 ABCA10_1 ABCA5_1 53 83 62 94 76 91 95 99 92 ABCE ABCA6_1 39 95 ABCF 23 87 93 20 44 ABCA1_1 I ABCA2_1 ABCA7_1 ABCA4_1 ABCA3_1 ABCA12_1 ABCA13 97 ABCA8_2 91 ABCA9_2 ABCA10_2 ABCA6_2 ABCA5_2 ABCA12_2 ABCA1_2 ABCA7_2 ABCA4_2 ABCA2_2 ABCA3_2 ABCA II 0.15 Figure 3.2 Phylogenetic tree of the human ABC genes Amino acid sequences containing ATP-bindingdomain proteins were identified with the model ABC_tran (accession PF00005) of the pfam database (Bateman et al., 1999) as described earlier (Dean et al., 2001) HUMAN AND DROSOPHILA ABC PROTEINS TABLE 3.2 DROSOPHILA ABC GENES Gene CG3156 CG2759 CG1703 CG1824 CG9281 CG8473 CG12703 CG1819 CG1718 CG1801 CG1494 CG3164 CG4822 CG17646 CG9892 CG9664 CG9663 CG3327 CG2969 CG11147 CG7806 CG7627 CG5853 CG5772 CG6214 CG7491 CG17338 CG10441 CG9270 CG8799 CG3879 CG8523 CG8908 CG10505 CG17632 CG7955 CG10226 Mdr65 CG5651 CG7346 CG4314 CG5944 CG6052 CG9330 CG14709 CG4225 CG4562 CG4794 CG5789 CG18633 CG11069 CG6162 CG9990 CG11898 CG11897 CG2316 Alias Size (aa) Family Location (Chr Nuc) Cyto Loc w 696 901 761 611 2556 618 1500 1713 1511 1197 620 643 627 615 609 812 729 832 705 1487 1327 689 2250 1896 324 1275 1307 1014 1344 1279 1313 1382 1283 755 606 1320 1302 611 597 666 1463 1660 708 1307 866 1348 711 1239 702 602 535 808 1302 1346 730 B G E B E A D A A A A G G G G G G G G H C C G C C A B B C C B B A C G B B B E G G A A E C B C A C G G H H C C D X 252038-254671 X 2545753-2539884 X 11393813-11396731 X 12363742-12360802 X 15454374-15450765 X 15513659-15523896 X 19494615-19497465 X 20757531-20763638 X 20909795-20902146 X 20924492-20917580 X 20896205-20901578 2L 123902-117541 2L 112000-116000 2L 1720498-1727693 2L 2649300-2658596 2L 3211844-3209624 2L 3214000-3220000 2L 3257267-325948 2L 4251813-4262480 2L 5656028-5653232 2L 8212839-8218079 2L 8262316-8256791 2L 9854119-9847658 2L 10105357-10089272 2L 12619174-12641593 2L 13675599-13676775 2L 18829742-18834099 2L 18835157-18839979 2L 20741821-20738317 2R 4426560-4431236 2R 7940090-7934079 2R 9235904-9241222 2R 15203694-15208725 2R 16226805-16222698 2R 18476505-18465883 3L1597621-1602155 3L 6180561-6175400 3L 6186691-6181468 3L 8895129-8892720 3L 11555624-11559309 3L 16398050-16400715 3L 17695681-17689489 3L 17627439-17622025 3L 1971540-1947231 3R 7362645-7369141 3R 11615803-11612420 3R 15626899-15619809 3R 15725586-15728807 3R 29281221-20277309 3R 29625526-29622829 3R 20635134-20637920 3R 22087630-22088417 3R 24409613-24429503 3R 24887241-24892598 3R 24881629-24885998 154260-145146 1B4 3B4 10C10 11B16 13E14 13E18–F1 18F1–F2 19F1 19F2 19F2 19F2 21B 21B 22B3 23A6 23E4–23E5 23E4–23E5 23F 24F8 26A1 29A3-A4 29B1 30E1–30E3 31A2 33F2 34D1 37B9 37B9 39A2 45D1 49E1 50F1 56F11 57D2 59E3 62B1 65A14 65A14 66E3–E4 68C10–C11 73A3 74E3–E4 74E3–E4 76B6 86F1 89A11–A12 92B9 92C1 96A7 96B5 96B6 97B1 98F1 99A 99A 101F Atet Sur Mdr49 Mdr50 bw st 51 52 ABC PROTEINS: FROM BACTERIA TO MAN 50 16 13 65 30 44 45 27 28 27 58 87 32 70 CG1 0226 Md-65 Md-49 ABCB1_1h ABCB1_2h Md-50 CG10226 Md-65 Md-49 Md-50 CG1824 CG4225 CG7955 CG3156 ABCB1 I and II 100 24 ABCD1h CG2316 CG7806 Sur 84 92 99 81 57 ABCE1h 100 68 ABCC1_1h CG6214 CG5789 CG11898 CG17338 34 CG10505 CG11897 29 CG9270 16 CG14709 53 CG10441 92 CG8799 69 CG4562 49 79 CG7627 63 ABCD ABCCI ABCE CG5051 CG5051 CG1484 CG7806 34 99 99 23 27 16 24 14 75 24 26 70 21 CG1801 ABCG CG11009 CG1718 CG5944 CG54794 CG8908 94 ABCF II CG18633 ABCA1_1h 24 20 14 ABCF I CG8473 CG1819 10 17 CG9281 CG9330 CG1703 99 Aet CG3164 CG5853 CG17646 CG9663 CG9892 CG4822 CG7346 CG9664 bw st CG3327 white 68 11 CG9281 CG9330 ABCG1h 88 ABCC II ABCF1_1h ABCF1_2h 58 19 50 CG1703 74 39 CG6214 CG11897 CG11898 CG10505 CG5789 41 60 CG17338 CG8799 40 CG7627 91 40 CG10441 CG9270 51 96 CG14709 80 CG4562 57 79 96 ABCC12h 78 89 76 CG7491 CG6162 CG9990 CG11147 ABCA I CG8473 ABCH CG1801 CG1819 ABCA1_2h 51 38 55 CG8909 CG1718 CG5944 CG1494 ABCA II 0.25 Figure 3.3 Phylogenetic tree of the Drosophila ABC genes Analysis (as described for Figure 3.2) was performed with all extracted Drosophila predicted protein sequences and a representative of each human subfamily N- and C-terminal ATP-binding domains of full transporters are included as separate units HUMAN AND DROSOPHILA ABC PROTEINS 14 15 16 17 18 19 20 21 10 22 11 X 12 13 A B C D E F G Y Figure 3.4 Map of human ABC genes A schematic map is shown for each human chromosome, with the approximate location of all ABC genes Clustered genes have a single line connecting to the chromosome The key indicates the subfamily for each gene (A ‫ ؍‬ABCA, etc.) X 2L 2R 3L 3R A B C D E F G H Figure 3.5 Map of Drosophila ABC genes A map is shown for each Drosophila chromosome with the approximate location of all identified ABC genes Clustered genes have a single line connecting to the chromosome The key indicates the subfamily for each gene (A ‫ ؍‬ABCA, etc.) independent and ancient gene duplication events Several Drosophila ABCB genes, Mdr49, Mdr50 and Mdr65, have been well characterized A fourth member of this group, CG10226, was identified as clustered with Mdr65 (Figure 3.5) All these genes are closely related to the human and mouse P-glycoproteins (ABCB1, ABCB4) and disruption of Mdr49 results in sensitivity to colchicines (Wu et al., 1991) Three genes, CG9990, CG6162 and CG11147, were identified that not fit into any of the known subfamilies and, in fact, are most closely related to ABC genes from bacteria (i.e Rhizobium NodI and E coli YhiH (subfamily NOD and DRI, respectively; see also Chapter 1) There are no close homologues to these genes in any other eukaryotic genome, including worms and plants The three genes are within large sequence contigs and have introns, therefore excluding the possibility of contamination from bacterial sequences In addition, this group forms a distinct cluster on the Drosophila tree Apart from the eye pigment precursor transporters white, scarlet and brown, very few Drosophila genes are associated with known 53 54 ABC PROTEINS: FROM BACTERIA TO MAN functions Knockout technology will have to be employed to begin to elucidate the functions of these genes In addition, very few Drosophila genes have clear orthologues in the human genome, suggesting consistent duplication and loss of ABC genes during the evolution of eukaryotic ABC genes HUMAN ABC GENE SUBFAMILIES ABCA (ABC1) This subfamily comprises 12 full transporters (Table 3.1), which are further divided into two subgroups based on phylogenetic analysis and intron structure (Arnould et al., 2001; Broccardo et al., 1999) The first group includes seven genes dispersed on six different chromosomes (ABCA1–A4, A7, A12, A13), whereas the second group contains five genes (ABCA5–A6, A8–A10) arranged in a cluster on chromosome 17q24 (Arnould et al., 2001) The ABCA subfamily contains some of the largest ABC genes, several of which encode over 2100 amino acids Representative examples of the major human ABC transporters, including ABCA4, are described in detail in several chapters in this volume The ABCA4 gene is expressed exclusively in photoreceptors, where it transports retinol (vitamin A) derivatives from the photoreceptor outer segment disks into the cytoplasm (Allikmets et al., 1997) The chromophore of a visual pigment rhodopsin, retinal, or its conjugates with phospholipids are the likely substrates for ABCA4, as they stimulate the ATP hydrolysis of the intact protein (Sun et al., 1999) Mice lacking Abca4 show increased levels of all-trans-retinaldehyde (all-trans-RAL) following light exposure, elevated phosphatidylethanolamine (PE) in outer segments, accumulation of the protonated Schiff base complex of all-trans-RAL and PE (N-retinylidene-PE), and striking deposition of a major lipofuscin fluorophore (A2-E) in retinal pigment epithelium (RPE) (Weng et al., 1999) These data suggest that ABCR is an outwardly directed flippase for N-retinylidene-PE Mutations in the ABCA4 gene have been associated with multiple eye disorders (Allikmets, 2000) A complete loss of ABCA4 function leads to retinitis pigmentosa whereas patients with at least one missense allele have Startgardt disease (STGD) STGD is characterized by juvenile to early adult onset of macular dystrophy with loss of central vision Carriers of the ABCA4 mutation also occur at increased frequency in age-related macular degeneration (AMD) patients AMD patients display a variety of phenotypic features, including the loss of central vision, after the age of 60 The causes of this complex trait are poorly understood, but a combination of genetic and environmental factors plays a role The abnormal accumulation of retinoids, due to ABCA4 deficiency, has been postulated to be one mechanism by which this process could be initiated Consistent with this hypothesis, mice heterozygous for Abca4 mutations accumulate lipofuscin-containing particles in their RPE cells (Mata et al., 2001) Tangier disease is characterized by deficient efflux of lipids from peripheral cells, such as macrophages, and a very low level of highdensity lipoproteins (HDL) The disease is caused by alterations in the ABCA1 gene, implicating this protein in the pathway of removal of cholesterol and phospholipids onto HDL particles (Young and Fielding, 1999) Patients with hypolipidemia have also been described who are heterozygous for ABCA1 mutations, suggesting that ABCA1 variations may play a role in regulating the level of HDLs in the blood (Marcil et al., 1999) ABCA1 gene expression is regulated by sterols (Langmann et al., 1999) and current models for ABCA1 function place it at the plasma membrane mediating the transfer of phospholipid and cholesterol onto lipid-poor apolipoproteins to form nascent HDL particles The ABCA1-mediated efflux of cholesterol is regulated by nuclear hormone receptors, such as oxysterol receptors (LXRs) and the bile acid receptor (FXR), which form heterodimers with retinoid X receptors (RXRs) (Repa et al., 2000) ABCB (MDR/TAP) The ABCB subfamily is unique in that it contains both full transporters and half transporters Four full transporters and seven half transporters have been currently described as members of this subfamily ABCB1 (MDR/ PGY1) was the first human ABC transporter cloned and characterized through its ability to confer a multidrug resistance phenotype to cancer cells (Juliano and Ling, 1976) ABCB1 was demonstrated to transport several hydrophobic substrates including drugs such as colchicine, VP16, HUMAN AND DROSOPHILA ABC PROTEINS adriamycin and vinblastine as well as lipids, steroids, xenobiotics and peptides (reviewed in Ambudkar and Gottesman, 1998) The gene is thought to play an important role in removing toxic metabolites from cells, and is also expressed in cells at the blood–brain barrier, where it plays a role in transporting into the brain compounds such as ivermectin and cortisol that cannot be delivered by diffusion ABCB1 also affects the pharmacology of drugs that are substrates, and a common polymorphism in the gene influences digoxin uptake (Hoffmeyer et al., 2000) Several ABC transporters are specifically expressed in the liver These play a role in the secretion of components of the bile, and are responsible for several forms of progressive familial intrahepatic cholestasis (PFIC), through intracellular accumulation of bile salts PFICs are a heterogeneous group of autosomal recessive liver disorders, characterized by early onset of cholestasis, which leads to liver cirrhosis and failure (Alonso et al., 1994) The ABCB4 (PGY3) gene transports phosphatidylcholine across the canalicular membrane of hepatocytes (van Helvoort et al., 1996) Mutations in this gene cause PFIC3, which results in a defect in the transport of phosphatidylcholine across the canalicular membrane of the hepatocyte (Deleuze et al., 1996; de Vree et al., 1998) PFIC3 is also associated with intrahepatic cholestasis of pregnancy (Dixon et al., 2000) The ABCB11 gene was originally identified based on homology to ABCB1 (Childs et al., 1995) ABCB11 is highly expressed on the liver canalicular membrane and has been demonstrated to be the major bile salt export pump Mutations in ABCB11 are found in patients with PFIC2, a disease associated with very low secretion of biliary bile salts (Strautnieks et al., 1998) The ABCB2 and ABCB3 (TAP) genes are half transporters that form heterodimers to transport into the ER peptides that are presented as antigens by the Class I HLA molecules The closest homologue of the TAPs, the ABCB9 half transporter, has been localized to lysosomes Several half transporters of the MDR/TAP subfamily have been localized to the inner membrane of the mitochondria The yeast orthologue of ABCB7, Atm1, has been implicated in mitochondrial iron homeostasis, as a transporter in the biogenesis of cytosolic Fe/S proteins (Kispal et al., 1997) Two distinct missense mutations in ABCB7 are associated with the X-linked anemia and ataxia (muscle non-coordination) (XLSA/A) phenotype (Allikmets et al., 1999) Three more half transporters from this subfamily, ABCB6, ABCB8 and ABCB10, have also been localized to mitochondria (Table 3.1) ABCC (CFTR/MRP) The ABCC subfamily contains 12 full transporters with diverse functional spectra including toxin secretion activities, ion transport, and regulation of a cell surface receptor The ABCC1 gene was identified in the small cell lung carcinoma cell line NCI-H69, a multidrug resistant cell that did not overexpress ABCB1 (Cole et al., 1992) The ABCC1 pump confers resistance to doxorubicin, daunorubicin, vincristine, colchicines and several other compounds, a very similar profile to that of ABCB1 However, unlike ABCB1, ABCC1 transports drugs that are conjugated to glutathione (Borst et al., 2000) ABCC1 can also transport leukotrienes such as leukotriene C4 (LTC4) LTC4 is an important signaling molecule for the migration of dendritic cells Migration of dendritic cells from the epidermis to lymphatic vessels is defective in Abcc1 Ϫ/Ϫ mice (Robbiani et al., 2000) ABCC2 and C3 also transport drugs conjugated to glutathione and other organic anions The ABCC4, C5, C11 and C12 proteins are smaller than the other MRP1-like genes and lack a proximal domain near the N-terminus (Borst et al., 2000), which is not essential for transport function (Bakos et al., 2000) The ABCC4 and C5 proteins confer resistance to nucleosides including the drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and purine analogues The rat Abcc2 gene was found to have a frameshift mutation in the strain defective in canalicular multispecific organic anion transport, the TRϪ rat (Paulusma et al., 1996) The TRϪ rat is an animal model of Dubin–Johnson syndrome and mutations in ABCC2 have been identified in Dubin–Johnson syndrome patients (Wada et al., 1998) The ABCC2 protein is expressed on the canalicular side of the hepatocyte and mediates organic anion transport, important for conjugation to and detoxification of many endogenous and xenobiotic lipophilic compounds in the liver Patients with Dubin–Johnson syndrome display hyperbilirubinemia, deposition of melanin-like pigment in liver cells, and in some cases, hepatomegaly and abdominal pain The CFTR/ABCC7 protein is a chloride ion channel that plays a role in all exocrine secretions, 55 56 ABC PROTEINS: FROM BACTERIA TO MAN and mutations in CFTR cause cystic fibrosis (Quinton, 1999) Cystic fibrosis is the most common fatal childhood disease in Caucasian populations, reaching frequencies ranging from 1/900 to 1/2500 The most common allele is a deletion of three base pairs (⌬F508) This allele is found in 85% of CF chromosomes in some populations, particularly northern Europeans At least two populations have a high frequency of other CF alleles The W1282X allele constitutes 51% of the CF alleles in the Ashkenazi Jewish population and the 1677delTA allele has been found at a high frequency in Georgians and is also present at an elevated level in Turkish and Bulgarian populations This has led several groups to hypothesize that these alleles arose through selection of an advantageous phenotype in the heterozygotes It is through CFTR as a surface receptor that some bacterial pathogens such as cholera and E coli cause increased fluid flow by release of toxins in the intestine, and resulting diarrhea Therefore several researchers have proposed that the CF mutations have been selected for in response to these disease(s) This hypothesis is supported by studies showing that indeed CF homozygotes fail to secrete chloride ions in response to a variety of stimulants, and a study in mice in which heterozygous null animals showed reduced intestinal fluid secretion in response to cholera toxin (Gabriel et al., 1993) CFTR is also the receptor for Salmonella typhimurium and is implicated in the innate immunity to Pseudomonas aeruginosa (Pier et al., 1998) Patients with two severe CFTR alleles such as ⌬F508 typically display severe disease with inadequate secretion of pancreatic enzymes (Quinton, 1999), leading to nutritional deficiencies, bacterial infections of the lung, and obstruction of the vas deferens, leading to male infertility Patients with at least one partially functional allele display enough residual pancreatic function to avoid the major nutritional and intestinal deficiencies (Dean et al., 1990), and subjects with very mild alleles display only congenital absence of the vas deferens with none of the other symptoms of CF Recently, heterozygotes of CF mutations have been found to have an increased frequency of pancreatitis (Cohn et al., 1998) and bronchiectasis (Pignatti et al., 1995) Thus, there is a spectrum of severity in the phenotypes caused by this gene that is inversely related to the level of CFTR activity Clearly other modifying genes and the environment also affect disease severity, particularly the pulmonary phenotypes The ABCC8 gene is a high-affinity receptor for the drug sulfonylurea Sulfonylureas are a class of drugs widely used to increase insulin secretion in patients with non-insulin-dependent diabetes These drugs bind to the ABCC8 protein and inhibit an associated potassium channel, under the control of ABCC8 Familial persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is an autosomal recessive disorder in which subjects display unregulated insulin secretion The disease was mapped to 11p15-p14 by linkage analysis, and mutations in the ABCC8 gene are found in PHHI families (Thomas et al., 1995) The ABCC8 gene has also been implicated in the insulin response in Mexican-American subjects (Goksel et al., 1998) and in type diabetes in French Canadians (Reis et al., 2000) but not in a Scandinavian cohort (Altshuler et al., 2000) ABCD (ALD) The ABCD subfamily contains four genes in the human genome and two each in the Drosophila and yeast genomes The yeast PXA1 and PXA2 products dimerize to form a functional transporter involved in very long chain fatty acid oxidation in the peroxisome (Shani and Valle, 1998) All yeast and human ABCD genes encode half transporters that are located in the peroxisome, where they function as homoand/or heterodimers in the regulation of very long chain fatty acid transport Adrenoleukodystrophy (ALD) is an X-linked recessive disorder characterized by neurodegenerative phenotypes with onset typically in late childhood and caused by mutations in the ABCD1 gene (Mosser et al., 1993) Adrenal deficiency commonly occurs and the presentation of ALD is highly variable Adrenomyeloneuropathy (AMN), childhood ALD and adultonset forms are recognized, but there is no apparent correlation to ABCD1 alleles ALD patients have an accumulation of unbranched saturated fatty acids with a chain length of 24 to 30 carbons, in the cholesterol esters of the brain and in adrenal cortex The ALD protein, like its yeast homologue, is located in the peroxisome, where it is believed to be involved in the transport of very long chain fatty acids ABCE (OABP) AND ABCF (GCN20) The ABCE and ABCF subfamilies contain genes that have ATP-binding domains that HUMAN AND DROSOPHILA ABC PROTEINS are clearly derived from ABC transporters but they have no TMD and are not known to be involved in any membrane transport functions The ABCE subfamily is composed solely of the oligoadenylate-binding protein, a molecule that recognizes oligoadenylate produced in response to infection by certain viruses This gene is found in multicellular eukaryotes but not in yeast, suggesting it is part of innate immunity Each ABCF gene contains a pair of NBDs The best-characterized member, the S cerevisiae GCN20 gene, mediates the activation of the eIF-2 alpha kinase (Marton et al., 1997) and a human homologue, ABCF1, is associated with the ribosome and appears to play a similar role (Tyzack et al., 2000) ABCG (WHITE) The human ABCG subfamily is composed of six half transporters that have an NBD at the N-terminus and a TMD at the C-terminus The most intensively studied ABCG gene is the white locus of Drosophila The white protein, together with brown and scarlet, transport precursors of eye pigments (guanine and tryptophan) in the eye cells of the fly (Chen et al., 1996) The mammalian ABCG1 gene is involved in cholesterol transport regulation (Klucken et al., 2000) Two half-transporter genes, ABCG5 and ABCG8, were identified (Berge et al., 2000; Lee et al., 2001; Shulenin et al., 2001), located headto-head on the human chromosome 2p15-p16, and regulated by the same promoter These genes are both mutated in families with sitosterolemia, a disorder characterized by defective transport of plant and fish sterols and cholesterol Most likely, the two half transporters form a functional heterodimer However, since ABCG5 is more frequently mutated in Asian and ABCG8 in Caucasian populations, they may also act as homodimers The ABCG1 gene is also regulated by cholesterol (Klucken et al., 2000) and ABCG3 is highly expressed in the liver, suggesting that these two genes may also be involved in cholesterol transport (Table 3.1) Analysis of cell lines resistant to mitoxantrone that not overexpress ABCB1 or ABCC1 led several laboratories to identify the ABCG2 (ABCP, MXR1, BCRP) gene as a drug transporter (Allikmets et al., 1998; Doyle et al., 1998; Miyake et al., 1999) ABCG2 confers resistance to anthracycline anticancer drugs and is amplified or involved in chromosomal translocations in cell lines selected with topotecan, mitoxantrone or doxorubicin treatment It is suspected that ABCG2 functions as a homodimer, because transfection of the gene into cells is sufficient to confer resistance to chemotherapeutic drugs ABCG2 can also transport several dyes such as Rhodamine and Hoechst 33462 and the gene is highly expressed in a subpopulation of hematopoietic stem cells (side population) that stain poorly for these dyes (Zhou et al., 2001) However, the normal function of the gene in these cells is unknown ABCG2 is highly expressed in the trophoblast cells of the placenta This suggests that the pump is responsible either for transporting compounds into the fetal blood supply, or for removing toxic metabolites The gene is also expressed in the intestine and inhibitors could be useful in making substrates orally available Other ABCG genes include ABCG3, to date exclusively found in rodents (Mickley et al., 2000), and the ABCG4 gene, which is expressed predominantly in human brain The functions of these two genes are unknown CONCLUSIONS AND PERSPECTIVES The complete identification of all the ABC genes in several eukaryotic genomes allows a comprehensive picture of the evolution of these genes to be ascertained Surprisingly, there are more ABC genes in both the Drosophila and C elegans genomes than there are in the human genome (Table 3.3) Although all of these species have the same seven ABC gene subfamilies, TABLE 3.3 ABC GENE SUBFAMILIES IN CHARACTERIZED EUKARYOTES Subfamily Human Drosophila C elegans Yeast A B C D E F G H Other Total 10 10 12 15 56 23 11 0 58 10 3a 31 a 12 11 12 0 48 Includes YDRO91c, YFL028c, YDR061w 57 58 ABC PROTEINS: FROM BACTERIA TO MAN and a fairly similar number of genes in each subfamily, it seems that most of these genes arose by unique gene duplication and deletion events Therefore, very few ABC genes have retained conserved functions throughout eukaryotic evolution The exceptions appear to be the organelle specific genes such as the ABCD genes found in the peroxisome and the ABCB genes in the mitochondria, all of which represent half transporters Identifying the function for ABC genes is a challenging task There is very little correlation between the gene sequence and the specificity of substrates identified to date For instance, ABCB1, ABCC1 and ABCG2 all 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ABCA1 ABCA2 ABCA3 ABCA4 ABCA5 ABCA6 ABCA7 ABCA8 ABCA9 ABCA10 ABCA12 ABCA 13 ABC1 ABC2 ABC3 , ABCC ABCR 9q31.1 9q34.4 16p 13. 3 1p21 .3 17q24 .3 17q24 .3 19p 13. 3 17q24 .3 17q24 .3 17q24 .3 2q34 7p12 .3 Ubiquitous... ABCC5_1 ABCC11_1 ABCC12_1 ABCD4 ABCD3 92 86 II ABC B11_2 ABCB5_1 ABCB11_1 ABCB1_1 ABCB4_1 ABCD1 ABCD2 100 ABCF1_1 I ABCF3_1 ABCF2_1 ABCF3_1 ABCF1_2 II ABCF2_2 ABCE1 ABCG2 ABCG8 ABCG5 ABCG ABCG1 ABCG4... 73 92 100 ABCB I ABCC7_2 ABCC10_2 ABCC11_2 ABCC12_2 ABCC5_2 100 ABCC8_2 ABCC9_2 ABCC4_2 ABCC2_2 ABCC1_2 ABCC3_2 ABCC10_1 ABCC4_1 ABCC7_1 ABCC6_1 100 ABCC8_1 ABCC9_1 ABCC1_1 ABCC2_1 ABCC3_1 ABCC5_1