The Human ATP-Binding Cassette (ABC) Transporter Superfamily

45 Pages·2001·0.537 MB·English

by  Dean M.

MichaelDean HumanABCTransporterSuperfamily
The Human ATP-Binding
Cassette (ABC) Transporter
Superfamily
by Michael Dean
Human Genetics Section, Laboratory of Genomic Diversity, National Cancer
Institute-Frederick. Correspondence to: Dr. Michael Dean, Bldg. 560, Room
21-18, NCI-Frederick, Frederick, MD 21702, USA. Telephone [phone];
Fax [phone];[email protected]
Abstract
The ATP-binding cassette (ABC) transporter superfamily contains membrane
proteins that translocate a wide variety of substrates across extra- and
intracellular membranes, including metabolic products, lipids and sterols,
and drugs. Overexpression of certain ABC transporters occurs in cancer cell
lines and tumors that are multidrug resistant. Genetic variation in these
genes is the cause or contributor to a wide variety of human disorders with
Mendelian and complex inheritance including cystic fibrosis, neurological
disease, retinal degeneration, cholesterol and bile transport defects,
anemia, and drug response phenotypes. Conservation of the ATP-binding
domains of these genes has allowed the identification of new members of
the superfamily based on nucleotide and protein sequence homology.
Phylogenetic analysis places the 48 known human ABC transporters into
seven distinct subfamilies of proteins. For each gene, the precise map
location on human chromosomes, expression data, and localization within
the superfamily have been determined. These data allow predictions to be
made as to potential function(s) or disease phenotype(s) associated with
each protein. Comparison of the human ABC superfamily to that of other
sequenced eukaryotes including Drosophila indicated that there is a rapid
rate of birth and death of ABC genes and that most members carry out
highly specific functions that are not conserved across distantly related
phyla.
Introduction to ABC Protein and Gene
Organization
TheATP-bindingcassette(ABC)genesrepresentthelargestfamilyoftransmembrane
(TM)proteins.TheseproteinsbindATPandusetheenergytodrivethetransportof
variousmoleculesacrossallcellmembranes(1–3)(Figure1).Proteinsareclassifiedas
ABCtransportersbasedonthesequenceandorganizationoftheirATP-bindingdomain
(s),alsoknownasnucleotide-bindingfolds(NBFs).TheNBFscontaincharacteristicmotifs
(WalkerAandB),separatedbyapproximately90–120aminoacids,foundinallATP-
bindingproteins(Figure1).ABCgenesalsocontainanadditionalelement,thesignature
(C)motif,locatedjustupstreamoftheWalkerBsite(4).Thefunctionalproteintypically
containstwoNBFsandtwoTMdomains(Figure2).TheTMdomainscontain6–11
membrane-spanningα-helicesandprovidethespecificityforthesubstrate.TheNBFsare
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locatedinthecytoplasmandtransfertheenergytotransportthesubstrateacrossthe
membrane.ABCpumpsaremostlyunidirectional.Inbacteria,theyarepredominantly
involvedintheimportofessentialcompoundsthatcannotbeobtainedbydiffusion
(sugars,vitamins,metalions,etc.)intothecell.Ineukaryotes,mostABCgenesmove
compoundsfromthecytoplasmtotheoutsideofthecellorintoanintracellular
compartment[endoplasmicreticulum(ER),mitochondria,peroxisome].Mostofthe
knownfunctionsofeukaryoticABCtransportersinvolvetheshuttlingofhydrophobic
compoundseitherwithinthecellaspartofametabolicprocessoroutsidethecellfor
transporttootherorgans,orforsecretionfromthebody.
Figure 1:DiagramofatypicalABCtransporterprotein.
A.AdiagramofthestructureofarepresentativeABCproteinisshownwithalipid bilayerinyellow,theTM
domainsin blue,andtheNBFinred.Althoughthemostcommonarrangementisafull transporterwithmotifs
arrangedN-TM-NBF-TM-NBF-C,asshown,NBF-TM-NBF-TM,TM-NBF,andNBF-TMarrangementsarealsofound.
B.TheNBFofanABCgenecontainstheWalkerAandBmotifsfound in allATP-bindingproteins.Inaddition,a
signatureorCmotifisalsopresent.Abovethediagramarethemostcommonaminoacidsfound inthesemotifs;
subfamiliesoftencontaincharacteristicresiduesintheseandotherregions.From(5).
Figure 2:ABCgenestructure.
AdiagramofanABChalf transporterandafull transporter.Thehalf transportercanformhomo-or
heterodimers,whereastheentirefull transporterisfound inonetranscript.
TheeukaryoticABCgenesareorganizedeitherasfulltransporterscontainingtwo
TMsandtwoNBFs,orashalftransporters(4)(Figure2).Thelattermustformeither
homodimersorheterodimerstoformafunctionaltransporter.ABCgenesarewidely
dispersedineukaryoticgenomesandarehighlyconservedbetweenspecies,indicating
thatmostofthesegeneshaveexistedsincethebeginningofeukaryoticevolution.The
genescanbedividedintosubfamiliesbasedonsimilarityingenestructure(halfversus
fulltransporters),orderofthedomains,andonsequencehomologyintheNBFandTM
domains.TherearesevenmammalianABCgenesubfamilies,fiveofwhicharefoundin
theSaccharomycescerevisiaegenome(5).
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AlistofWebresourcesonABCgenesandproductscanbefoundinBox1.
AmoredetailedaccountofeachofthehumanABCgenes[http://www.ncbi.nlm.nih.
gov/cgi-bin/Entrez/map_search?chr=hum_chr.inf&query=ATP-binding
+cassette&qchr=&advsrch=off]isgivenbelow.Foreachgene,aconcisedescriptionis
givenontheknownfunctionanddiseaseinvolvement,andlinkstootherdatabases,such
asUniGene,OMIM,andGenBank,aregivenwhereappropriate.Thisisacomprehensive
treatment:evengenesthatareverypoorlycharacterizedareincluded.Forgenessuchas
CFTRandABCB1/PGP/MDRthathavebeenstudiedextensively,abriefreviewisgiven
withlinkstootherresourcesandreviewarticles.Suggestedcorrectionsandadditionsare
welcomeforfutureupdatesofthesepagesandshouldbesenttotheauthor(dean@ncifcrf.
gov).
Nomenclature
AllhumanandmouseABCgeneshavestandardnomenclature,developedbytheHuman
GenomeOrganization(HUGO)atameetingofABCgeneresearchers.Detailsofthe
nomenclatureschemecanbefoundat:http://www.gene.ucl.ac.uk/nomenclature/
genefamily/abc.html.
ResearchersworkingonABCC7/CFTR,ABCB2/TAP1,andABCB3/TAP2have
petitionedtokeeptheiroriginalgenedesignations.Officialgenesymbolsareusedinthis
monograph,butallknownsynonymsarealsoincludedtoallowresearcherstorefertothe
literature.
Overview of Human ABC Gene Subfamilies
AlistofallknownhumanABCgenesisdisplayedinTable1.Thislistincludesan
analysisofthereleasedgenomesequences(6,7).Ananalysisofthegenomesequence
indicatesthepresenceofatleast19pseudogenes(Dean,unpublished).Thereremain
severalsequencesinthegenomewithhomologytoABCgenesthatlieinincompletely
sequencedregionsandmayrepresentadditionalpseudogenesorfunctionalloci.
Table1.ListofhumanABCgenes,chromosomallocation,andfunction.
Symbol Alias Location Function
ABCA1 ABC1 9q31.1 CholesteroleffluxontoHDL
ABCA2 ABC2 9q34.3 Drugresistance
ABCA3 ABC3,ABCC 16p13.3 Surfactantsecretion?
ABCA4 ABCR 1p21.3 N-Retinylidiene-PEefflux
ABCA5 17q24.3
ABCA6 17q24.3
ABCA7 19p13.3
ABCA8 17q24.3
ABCA9 17q24.3
ABCA10 17q24.3
ABCA12 2q34
ABCA13 7p12.3
ABCB1 PGY1,MDR 7q21.12 Multidrugresistance
ABCB2 TAP1 6p21.3 Peptidetransport
ABCB3 TAP2 6p21.3 Peptidetransport
ABCB4 PGY3 7q21.12 PCtransport
ABCB5 7p21.1
ABCB6 MTABC3 2q35 Irontransport
ABCB7 ABC7 Xq21-q22 Fe/Sclustertransport
ABCB8 MABC1 7q36.1
ABCB9 12q24.31
ABCB10 MTABC2 1q42.13
ABCB11 SPGP 2q24.3 Bilesalttransport
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Symbol Alias Location Function
ABCC1 MRP1 16p13.12 Drugresistance
ABCC2 MRP2 10q24.2 Organicanionefflux
ABCC3 MRP3 17q21.33 Drugresistance
ABCC4 MRP4 13q32.1 Nucleosidetransport
ABCC5 MRP5 3q27.1 Nucleosidetransport
ABCC6 MRP6 16p13.12
CFTR ABCC7 7q31.31 Chlorideionchannel
ABCC8 SUR 11p15.1 Sulfonylureareceptor
ABCC9 SUR2 12p12.1 K(ATP)channelregulation
ABCC10 MRP7 6p21.1
ABCC11 16q12.1
ABCC12 16q12.1
ABCD1 ALD Xq28 VLCFAtransportregulation
ABCD2 ALDL1,ALDR 12q11
ABCD3 PXMP1,PMP70 1p22.1
ABCD4 PMP69,P70R 14q24.3
ABCE1 OABP,RNS4I 4q31.31 Oligoadenylatebindingprotein
ABCF1 ABC50 6p21.1
ABCF2 7q36.1
ABCF3 3q27.1
ABCG1 ABC8,White 21q22.3 Cholesteroltransport?
ABCG2 ABCP,MXR,BCRP 4q22 Toxinefflux,drugresistance
ABCG4 White2 11q23
ABCG5 White3 2p21 Steroltransport
ABCG8 2p21 Steroltransport
ByaligningtheaminoacidsequencesoftheNBFdomainsandperforming
phylogeneticanalysiswithanumberofmethods,theexistingeukaryoticgenescanbe
groupedintosevenmajorsubfamilies.Afewgenesdonotfitintothesesubfamilies,and
severalofthesubfamiliescanbefurtherdividedintosubgroups.
ABCA (ABC1)
ThehumanABCAsubfamilycomprises12fulltransporters(Table1)thatarefurther
dividedintotwosubgroupsbasedonphylogeneticanalysisandintronstructure(8,9).
Thefirstgroupincludessevengenesdispersedonsixdifferentchromosomes(ABCA1,
ABCA2,ABCA3,ABCA4,ABCA7,ABCA12,ABCA13),whereasthesecondgroupcontains
fivegenes(ABCA5,ABCA6,ABCA8,ABCA9,ABCA10)arrangedinaclusteron
chromosome17q24.TheABCAsubfamilycontainssomeofthelargestABCgenes,several
ofwhichareover2,100aminoacidslong.Twomembersofthissubfamily,theABCA1
andABCA4(ABCR)proteins,havebeenstudiedextensively.TheABCA1proteinis
involvedindisordersofcholesteroltransportandHDLbiosynthesis(seebelow).The
ABCA4proteintransportsvitaminAderivativesintheoutersegmentsofrod
photoreceptorcellsandthereforeperformsacrucialstepinthevisioncycle.
TheABCAgenesarenotpresentinyeast;however,evolutionarystudiesofABCA
genesindicatethattheyaroseashalftransportersthatsubsequentlyduplicated,andthat
certainsetsofABCAgeneswerelostindifferenteukaryoticlineages(10).
ABCB (MDR/TAP)
TheABCBsubfamilyisuniqueinmammalsinthatitcontainsbothfulltransportersand
halftransporters.Fourfulltransportersandsevenhalftransportershavecurrentlybeen
describedasmembersofthissubfamily.ABCB1(MDR/PGY1)isthefirsthumanABC
transporterclonedandcharacterizedthroughitsabilitytoconferaMDRphenotypeto
cancercells.ThephysiologicalfunctionalsitesofABCB1includetheblood-brainbarrier
andtheliver.TheABCB4andABCB11proteinsarebothlocatedintheliverandare
involvedinthesecretionofbileacids.TheABCB2andABCB3(TAP)genesarehalf
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transportersthatformaheterodimertotransportpeptidesintotheERthatarepresented
asantigensbytheclassIHLAmolecules.TheclosesthomologoftheTAPs,theABCB9
halftransporter,hasbeenlocalizedtolysosomes.Theremainingfourhalftransporters,
ABCB6,ABCB7,ABCB8,andABCB10,localizetothemitochondria,wheretheyfunction
inironmetabolismandtransportofFe/Sproteinprecursors.
ABCC (CFTR/MRP)
TheABCCsubfamilycontains12fulltransporterswithadiversefunctionalspectrumthat
includesiontransport,cell-surfacereceptor,andtoxinsecretionactivities.TheCFTR
proteinisachlorideionchannelthatplaysaroleinallexocrinesecretions;mutationsin
CFTRcausecysticfibrosis(11).ABCC8andABCC9proteinsbindsulfonylureaand
regulatepotassiumchannelsinvolvedinmodulatinginsulinsecretion.Therestofthe
subfamilyiscomposedofnineMRP-relatedgenes.Ofthese,ABCC1,ABCC2,andABCC3
transportdrugconjugatestoglutathionineandotherorganicanions.TheABCC4,ABCC5,
ABCC11,andABCC12proteinsaresmallerthantheotherMRP1-likegeneproductsand
lackanN-terminaldomain(12)thatisnotessentialfortransportfunction(13).The
ABCC4andABCC5proteinsconferresistancetonucleosidesincludingPMEAandpurine
analogs.ThehumangenomecontainsaseeminglyintactABCCgeneonchromosome21
(ABCCxP1)thatcontainsaframeshiftinoneexonandisthereforeapseudogene.The
sameframeshiftmutationispresentinthegorillaandchimpanzeehomologs,butthe
geneappearstobefunctionalandexpressedinmonkeys(Anniloetal.,inpreparation).
ABCD (ALD)
TheABCDsubfamilycontainsfourgenesinthehumangenomeandtwoeachinthe
Drosophilamelanogasterandyeastgenomes.TheyeastPXA1andPXA2productsdimerize
toformafunctionaltransporterinvolvedinverylongchainfattyacidoxidationinthe
peroxisome(14).Allofthegenesencodehalftransportersthatarelocatedinthe
peroxisome,wheretheyfunctionashomo-and/orheterodimersintheregulationofvery
longchainfattyacidtransport.
ABCE (OABP) and ABCF (GCN20)
TheABCEandABCFsubfamiliescontaingeneproductsthathaveATP-bindingdomains
thatareclearlyderivedfromABCtransportersbuttheyhavenoTMdomainandarenot
knowntobeinvolvedinanymembranetransportfunctions.TheABCEsubfamilyis
solelycomposedoftheoligo-adenylate-bindingprotein,amoleculethatrecognizesoligo-
adenylateandisproducedinresponsetoinfectionbycertainviruses.Thisgeneisfound
inmulticellular eukaryotesbutnotinyeast,suggestingthatitispartofinnateimmunity.
EachABCFgenecontainsapairofNBFs.Thebest-characterizedmember,theS.
cerevisiaeGCN20geneproduct,mediatestheactivationoftheeIF-2αkinase(15),anda
humanhomolog,ABCF1,isassociatedwiththeribosomeandappearstoplayasimilar
role(16).
ABCG (White)
ThehumanABCGsubfamilyiscomposedofsix“reverse”halftransportersthathavean
NBFattheNterminusandaTMdomainattheCterminus.Themostintensivelystudied
ABCGgeneisthewhitelocusofDrosophila.Thewhiteprotein,alongwithbrownand
scarlet,transportsprecursorsofeyepigments(guanineandtryptophan)intheeyecellsof
thefly(17).ThemammalianABCG1proteinisinvolvedincholesteroltransport
regulation(18).OtherABCGgenesincludeABCG2,adrug-resistancegene;ABCG5and
ABCG8,codingfortransportersofsterolsintheintestineandliver;ABCG3,todate
exclusivelyfoundinrodents;andtheABCG4genethatisexpressedpredominantlyinthe
liver.Thefunctionsofthelasttwogenesareunknown.
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ABC Genes and Human Genetic Disease
ManyABCgeneswereoriginallydiscoveredduringthepositionalcloningofhuman
geneticdiseasegenes.Todate,14ABCgeneshavebeenlinkedtodisordersdisplaying
Mendelianinheritance(19)(Table2).Asexpectedfromthediversefunctionalrolesof
ABCgenes,thegeneticdeficienciesthattheycausealsovarywidely.BecauseABCgenes
typicallyencodestructuralproteins,allofthedisordersarerecessiveorX-linkedrecessive
andareattributabletoaseverereductionorlackoffunctionoftheprotein.However,
heterozygousvariantsinABCgenemutationsarebeingimplicatedinthesusceptibilityto
specificcomplexdisorders.
Table2.DiseasesandphenotyescausedbyABCgenes.
Gene Mendeliandisorder Complexdisease OMIM
ABCA1 Tangierdisease,FHDLDa 600046
ABCA4 Stargardt/FFM,RP,CRD,CD AMD 248200
ABCB1 Ivermectinsusceptibility Digoxinuptake 171050
ABCB2 Immunedeficiency 170260
ABCB3 Immunedeficiency 170261
ABCB4 PFIC3 ICP 171060
ABCB7 XLSA/A 300135
ABCB11 PFIC2 603201
ABCC2 Dubin-JohnsonSyndrome 601107
ABCC6 Pseudoxanthomaelasticum 603234
ABCC7 CysticFibrosis,CBAVD Pancreatitis,bronchiectasis 602421
ABCC8 FPHHI 600509
ABCD1 ALD 300100
ABCG5 Sitosterolemia 605459
ABCG8 Sitosterolemia 605460
aFHDLD,familialhypoapoproteinemia;FFM,fundusflavimaculatis;RP,retinitispigmentosum19;CRD,cone-
roddystrophy;AMD,age-relatedmaculardegeneration;PFIC,progressivefamilialintrahepaticcholestasis;ICP,
intrahepaticcholestasisofpregnancy;XLSA/A,X-linkedsideroblastosisandanemia;CBAVD,congential
bilateralabsenceofthevasdeferens;FPHHI,Familialpersistenthyperinsulinemichypoglycemiaofinfancy;
ALD,adrenoleukodystrophy.
FewABCgenemutationsarelethal.Untreatedcysticfibrosis(ABCC7/CFTR)is
typicallylethalinthefirstdecade,andadrenoleukodystrophy(ABCD1/ALD)canalsobe
fatalinthefirst10yearsoflife.TheonlymutationsdescribedinABCB7aremissense
alleles,andtheyeasthomologisessentialtomitochondria,suggestingthatthisgeneis
essential.TheonlydevelopmentaldefectascribedtoanABCgeneisthecongenital
absenceofthevasdeferensthatoccursinbothcysticfibrosispatientsandpatientswith
lesssevereallelesthatpresentmalesterilityastheironlyphenotype.Thus,mostABC
genesdonotplayanessentialroleindevelopment.
Mouse Knockouts
Mostofthehumangeneshaveaclearmouseortholog;however,thereareseveral
exceptions(Table3).SeveralABCgeneshavebeendisruptedinthemouse(Table3).
Theseincludesomeofthegenesmutatedinhumandiseases,aswellasseveralofthe
knowndrugtransporters.TheAbca1andCftr–/–miceshowcompromisedviability;
however,theremainingknockoutsareviableandfertile,andmanyshoweitherno
phenotypeoraphenotypeonlyunderstressedconditions.
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Table3.ABCgenes:humanandmouseorthologs.
Humangene Mousegene Locationa Knockout Reference
ABCA1 Abca1 4,23.1cM Yb Orso2000;McNeish2000
ABCA2 Abca2 2,12.6 N
ABCA3 Abca3 Unknown N
ABCA4 Abca4 3,61.8 Y Weng1999
ABCA5 Abca5 Unknown N
ABCA6 Abca6 Unknown N
ABCA7 Abca7 10,44 N
ABCA8 Abca8a Unknown N
Abca8b 11,69 N
ABCA9 Abca9 Unknown N
ABCA10
ABCA12 Abca12 1C1
ABCA13 Abca13 11A1
ABCB1 Abcb1a 5,1 Yc Schinkel1994
Abcb1b 5,1 Y Schinkel1997
ABCB2 Abcb2(Tap1) 17 Y VanKaer1992
ABCB3 Abcb3(Tap2) 17 N
ABCB4 Abcb4 5,1 Y Smit1993
ABCB5 Abcb5 12,60 Dean,etal.,unpublished
ABCB6 Abcb6 1,C3 N
ABCB7 Abcb7 X,39 N
ABCB8 Abcb8 Unknown N
ABCB9 Abcb9 5,F N
ABCB10 Abcb10 8,67 N
ABCB11 Abcb11 2,39 N
ABCC1 Abcc1 16 Y Lorico1997;Wijnholds1997
ABCC2 Abcc2 19 Yd Paulusma1996
ABCC3 Abcc3 Unknown N
ABCC4 Abcc4 13,E4 Dean,etal.,unpublished
ABCC5 Abcc5 16,14 N
ABCC6 Abcc6 7,B3 N
ABCC7 Abcc7(Cftr) 6,3.1 Y Dorin1992;Snouwaert1992;van
Doorninck1995
ABCC8 Abcc8 7,41 N
ABCC9 Abcc9 6,70 N
ABCC10 Abcc10 Unknown N
ABCC11 Abcc11 8,44-45
ABCC12
ABCD1 Abcd1 X,29.5 Y Forss-Petter1997
ABCD2 Abcd2 15,E-F N
ABCD3 Abcd3 3,56.6 N
ABCD4 Abcd4 12,39 N
ABCE1 Abce1 8,36 N
ABCF1 Abcf1 17,20.5 N
ABCF2 Abcf2 13,40 N
ABCF3 Abcf3 16,22 N
ABCG1 Abcg1 17,A2-B N
ABCG2 Abcg2 6,28.5 Y SorrentinoandSchinkel,unpublished
Abcg3 5,59 N
ABCG4 Abcg4 9,syntenic N
ABCG5 Abcg5 17,syntenic N
ABCG8 Abcg8 17,syntenic N
aThechromosomelocationofthegeneinthemouseisgivenalongwitheitherthedistancefromthecentromere
incentimorgansorthecytogeneticlocation.
bTheWHAMchicken(amodelofTangierdisease)(46)issuspectedofbeingmutantinAbca1.
cAbcb1mutantdogshavebeendescribed(84).
dAbcc2mutantratshavebeendescribed(132).
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Multidrug Resistance and Cancer Therapy
Cellsexposedtotoxiccompoundscandevelopresistancebyanumberofmechanisms
includingdecreaseduptake,increaseddetoxification,alterationoftargetproteins,or
increasedexcretion.Severalofthesepathwayscanleadtomultidrugresistance(MDR)in
whichthecellisresistanttoseveraldrugsinadditiontotheinitialcompound.Thisisa
particularlimitationtocancerchemotherapy,andtheMDRcelloftendisplaysother
properties,suchasgenomeinstabilityandlossofcheckpointcontrol,thatcomplicate
furthertherapy.ABCgenesplayanimportantroleinMDR,andatleastsixgenesare
associatedwithdrugtransport.
ThreeABCgenesappeartoaccountfornearlyalloftheMDRtumorcellsinboth
humanandrodentcells.TheseareABCB1/PGP/MDR1,ABCC1/MRP1,andABCG2/MXR/
BCRP(Table4).Noothergeneshavebeenfoundoverexpressedincellsthatdisplay
resistancetoawidevarietyofdrugsandincellsfrommicewithdisruptedAbcb1a,
Abcb1b,andAbcc1genes;theAbcg2genewasoverexpressedinallMDRcelllinesderived
fromavarietyofselections(20).
Table4.ABCtransportersinvolvedindrugresistance.
Gene Substrates Inhibitors
ABCB1 Colchicine,doxorubicin,VP16,aAdriamycin, Verapamil,PSC833,GG918,V-104,
vinblastine,digoxin,saquinivir,paclitaxel PluronicL61
ABCC1 Doxorubicin,daunorubicin,vincristine,VP16, CyclosporinA,V-104
colchicines,VP16,rhodamine
ABCC2 Vinblastine,sulfinpyrazone
ABCC3 Methotrexate,VP16
ABCC4 Nucleosidemonophosphates
ABCC5 Nucleosidemonophosphates
ABCG2 Mitoxantrone,topotecan,doxorubicin, FumitremorginC,GF120918
daunorubicin,CPT-11,rhodamine
aVP16,etoposide.
InhibitorsofthemajorABCgenescontributingtoMDRhavebeendeveloped,and
extensiveexperimentationandclinicalresearchhavebeenperformedtoattempttoblock
thedevelopmentofdrugresistanceduringchemotherapy(Table4).Thelatest
experimentswithhigh-affinityandhigh-specificityABCB1inhibitorsshowthatthegene
isexpressedinmanyprimarytumorsinhumanpatientsandthatitsactivitycanbe
blockedwithdosesofinhibitorthatdonothaveadversesideeffectsordisruptthe
pharmacologyofthedrugregimen(21).Thus,thedevelopmentofhighlyspecific
inhibitorstotheothermajordrugtransporterscouldleadtothedevelopmentofmuch
moreeffectivechemotherapyprotocols.
Anotherlimitationofchemotherapyisthenarrowdifferenceinsensitivityofthe
tumorcellstodrugsandsensitivityofthepatient'snormalstemcells.ABCgeneshave
alsobeenusedastoolstodeliverdrugtransporterstoearlystemcellsandtoprotectthem
fromchemotherapeuticdrugs.Thisstrategywouldallowhighdosesofdrugtobegiven
forlongerperiodsoftime.
Phylogenetic Analysis of Human ABC Genes
TheidentificationofthecompletesetofhumanABCgenesallowsacomprehensive
phylogeneticanalysisofthesuperfamily.AlignmentoftheNBFsfromeachgeneanda
neighbor-joiningtreeresultingfromthisanalysisisdisplayed(Figure3).The
subclassificationofABCtransportersisinexcellentagreementwiththephylogenetictrees
obtained.Inparticular,allmajorABCtransporterfamiliesarerepresentedinthehuman
treebystableclusterswithhighstatisticalsignificance.
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Figure 3:PhylogenetictreeofthehumanABCgenes.
ATP-bindingdomainproteinswereidentifiedusing themodelABC_tranofthePfamdatabase(250).The
HMMSEARCH programfromtheHMMERpackage(251)andasetofcustom-madeservicescriptswereusedto
extractATP-bindingdomainsfromallproteinsequencesofinterest.Notethatsomeproteinsanalyzedcontain
twoATP-bindingdomains(IandII),whereasotherscontainedonly oneATP-bindingdomain.Alignmentswere
generatedwiththehidden Markovmodel-basedHMMALIGNprogram(252)using theABC_tranmodel.The
resulting multiplealignmentwasanalyzedwithNJBOOT(N.Takezaki,personalcommunication),implementing
theneighbor-joiningtree-makingalgorithm(253);thenumberatthebranchofthenodesrepresentsthevalue
from100replications.Thedistancemeasurebetweensequencesusedfortree-makingwasthePoisson
correctionformultiplehits(254).Toverifytheposition ofthepreviously unknown subgroup ofDrosophilagenes
(CG6162,CG9990,andCG11147),thegeneswerealigned witharepresentativeofeachofthehuman
subfamilies.BecausesomeofthehumanproteinshadtwoATP-bindingdomains,thesetcontainedthree
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Drosophilaand12humansequences.TheJTTmodel(255),asdefined in theMOLPHYpackagewiththe“star
decomposition”option,wasused.Thetentativebesttree(thetotalnumberofpossibletreesfor15sequencesis
toolargeforexhaustivesearchthrough allofthesetrees)wasthenusedforlocalmaximumlikelihoodsearch
through thesurroundingtreetopologies.From(5).
ThisanalysisprovidescompellingevidenceforfrequentdomainduplicationofATP-
bindingdomainsinABCtransporters.Virtuallyinvariably,bothATP-bindingdomains
withinagenearemorecloselyrelatedtoeachotherthantoATP-bindingdomainsfrom
ABCtransportergenesofothersubfamilies.Thiscouldrepresentaconcertedevolutionof
domainswithinthesamegene,butthisseemsunlikelybecausethetwodomainswithin
eachgenearesubstantiallydiverged.Therefore,itappearsthatduplicationofATP-
bindingdomainswithinmajorABCfamilieswasaresultofseveralindependent
duplicationeventsratherthanasingleancestralduplication.
Mouse ABC Genes
AnalysisoftheCeleraassemblyofthemousegenomewasusedtoidentifyhomologsof
thehumanABCgenes.Withonlyafewexceptions,thereisconcordancebetweenthetwo
mammalianspecies(Table3).TheexceptionsareaduplicatedcopyoftheABCB1/PGP/
MDRgene(Mdr1b),anABCGfamilygenerelatedtoABCG2thatispresentinthemouse
andnotinthehuman(Abcg3)(22),lossofAbcc11(Dean,unpublished),duplicationofthe
ABCA8geneinthemouse(Abca8a),andalossinthemouseofABCA10(Anniloetal.,
submitted).Inaddition,micehaveaclusterofthreeABCAfamilygenesthatisnot
characterizedinthehumangenome(Chen,Annilo,Shulenin,andDean,unpublished).
Thisregionofthehumangenomeisincompletelycharacterizedanddoesnotcurrently
containanydescribedfunctionalloci.Therefore,micehave52ABCgenesandmostofthe
humangeneshaveasinglehomologinthemousegenome,indicatingthatthefunctions
ofthemousegenesshouldbehighlysimilartohumangenes.
Drosophila ABC Genes
TheorganizationandannotationoftheDrosophilaABCgeneshavebeendetermined
fromtheCelera(23)andFlybase(5)databases.Initialsubfamilyclassificationswere
assignedbasedonhomologyandBLASTscores,andthelocationofeachgeneisshown
(Table5).Intotal,thereare56geneswithatleastonerepresentativeofeachoftheknown
mammaliansubfamilies(Table6).Thesubfamilygroupingswereconfirmedby
phylogeneticanalyses.ArepresentativetreeisshowninFigure4.Asexpected,genes
fromthesamesubfamilyclustertogetherandconfirmtheinitialassignmentsmadeby
inspection.
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