52 OLEROFONECHAPERTEINSOPRINAHRFUNCTION this sensitizationfectefhas been observed with other AHR cess, under both basal and ligand-stimulated conditions ligands, a study on the fectef of XAP2 in the context of [33,60,65,69].Theseationsobservsupporttheconceptthat ferentdif classes of ligands, for example, antagonists and XAP2can alter AHR-mediatedtranscription.First,bylim- elnovveselectiAHRligands,hasyettobeundertaken.The itingtranslocation,XAP2canbeedclassi(cid:147)asaninhibitorof increasedligandvenessresponsiinthepresenceofXAP2has AHR action.,verselyContheutionredistribofunstimulated been edattribut to enhanced receptor stability rather than a AHRfromthenuclearcompartmentintothecytoplasmwill changeinreceptorconformation.Thishypothesis wassub- resultinatedveleoplasmiccytvelsleofAHR,whichislikely sequentlycon(cid:147)rmedinmammaliansystems,whichdemon- to increase the ligand vitysensitiin anyvengicell. Despite strated higher svelle of cytoplasmic AHR in COS-1 cells theseferencesdifofinterpretation,XAP2clearlyin(cid:148)uences transfected with XAP2 when compared to cells not trans- the localization of the AHR and a number of potential fectedwithXAP2[12,59,62].Furthermore,theincreasein mechanisms ehav been proposed to explain this phenome- AHR protein elicited by XAP2 was demonstrated to be non. The immunophilins present in the glucocorticoid speci(cid:147)c for XAP2, since immunophilins such as FKBP52 receptor ehav also been wnsho to in(cid:148)uence receptor local- failed to mediate a vepositi in(cid:148)uence on AHR svelle [46]. ization and transport through a mechanism that vesvolin Datastexithat discredit the notion that XAP2 can increase the peptidylprolyl isomerase domain of FKBP52, forming ligandvenessresponsithroughaconformationalchange[63]. a transport complex with microtubules to facilitate TP-A AHR ligand ssveneresponsi has been wnsho to exhibit a dependent nuclear utionredistrib [71]. A complementary marked degree of species ,dependency with mouse AHR mechanism volvingin XAP2 and AHR has largely been beingvefold(cid:147)morevesensitithanitshumanhomologtomost discounted,duetotheationobservthatchemicalmicrotubule butnotallAHRagonists[25,64].,verweHochimericmouse/ disruption fails to ertlyov in(cid:148)uence ligand-induced AHR humanreceptorsfailtoexchangeligand,vitysensitisuggest- translocation [33]. It has also been speculated that binding ingthatligandvenessresponsiisinherenttothereceptoritself of XAP2 to the AHR cytoplasmic xcomple may result in ratherthanaconsequenceofconformationalchangesfacil- physicalmaskingoraconformationalchangeofthenuclear itated by the XAP2 chaperone complex [63, 65]. Interest- localization signal, thus ventingpre translocation. Such a ,inglytheinitialpuri(cid:147)cationofAHRproteinwashamperedin mechanism seems ,unlikely since an antibody directed partbyitsinstabilityandacutevitysensititoheatstress.This against the nuclear localization signal is not blocked when vitysensiti to temperature can be whatsome ercomeov by AHRisboundtoXAP2[33].ranslocationToflargemolec- incorporationofXAP2intotheAHRcorecomplex,support- ular weight and multimeric escomplex containing nuclear ing a role of XAP2 in stabilizing the AHR complex [62]. localization signals across the nuclear opevelen is highly Furthervestigationinhasindicatedthatthestabilizingfectef regulated, vingvolin multiple adaptor proteins [72]. The ofXAP2,incombinationwithHSP90andp23,maybedueto AHRcomplexhasbeenwnshotointeractwithcomponents XAP2protectingAHRfromproteolyticadationdegrthrough of this transport machinery and suggest a mechanism by inhibitionofAHRubiquitinationbyE3ligases,suchasthat which XAP2 can in(cid:148)uence AHR compartmentalization observed with carboxyl terminus of Hsc70-interacting pro- [33, 73]. Immunoprecipitation and GST npull-dow studies tein(CHIP),althoughtheroleofCHIPinAHRadationdegr using the mouse AHR demonstrate a physical interaction has only been demonstrated inan in vitro context[66(cid:133)68]. with importin b , a member of the karyopherin family of In addition to stabilizing the AHR, studies utilizing adaptor proteins. Importin a interacts with basic arginine- (cid:148)uorescently tagged AHR identi(cid:147)ed XAP2 as a factor in- rich nuclear localization signals such as that carried by the vedvol in the cytoplasmic retention of the AHR. Although AHR.Subsequentinteractionofthiscomplexwithimportin viouslypre considered primarily cytoplasmic in the unli- b andGDP-boundRanfacilitatestransportacrossthenuclear gandedstate,theAHRhasactuallybeenwnshotoundergo velope.enInclusionofXAP2intheAHRcomplexappearsto dynamic nucleocytoplasmic shuttling in the absence of a diminishtheassociationofAHR,throughitsnuclearlocal- ligand,resultinginafusedifoplasmiccytandnuclearstaining ization signal, with importin b and thus is likely to limit whenvisualizedbyimmuno(cid:148)uorescenceortheuseof(cid:148)uo- transportintothenucleus,whichwouldexplaintheobserved rescently tagged AHR constructs [33, 69]. reatmentT with cytoplasmicretentionofAHR[33].ThenatureoftheXAP2- AHR ligands, for example, TCDD, prompts a marked and mediated inhibition of importin b binding has yet to be rapidionredistributofAHRintothenucleus,typicallywithin elucidated, ,but as mentioned ,earlier it does not seem to 1h, consistent with its role as a transcription .factor Intra- evolvinblockade ofthe nuclear localization sequence. cellularementmovoftheAHRthroughveconstitutinucleo- In conclusion, XAP2 represents a whatsome enigmatic oplasmiccyt shuttling or ligand-induced translocation is regulator of AHR function. First, XAP2 increases AHR broughtaboutthroughtheactionofastrongbipartitenuclear protein ,stability prompting an apparent enhancement of localization and export signals within the AHR [29, 70]. ligand,vitysensitiwhich leads to increased AHR-mediated XAP2xpressionereovstudiesinCOS-1cellsehavwnshothat transcriptionofappropriatetargetgeneswingfolloexposure XAP2 can markedly diminish the AHR translocation pro- toAHRagonists.,verweHoincorporationofXAP2intothe NCOMPOSITIOOFTHEDEDUNLIGANAHRCORECOMPLEX 53 TABLE3.1 XAP2-InteractingProteins ProteinsReportedtoInteractwithXAP2 Protein Comments References HepatitisBvirusXprotein InteractionwithXAP2identi(cid:147)edthroughyeasttwo-hybrid.assayXAP2 [76(cid:133)78,80,81] inhibitsationvtransactipotentialofHBxandmayin(cid:148)uencehepatitisB pathology Heatshockprotein90 XAP2undergoesevcooperatibindingtobothAHRandHSP90toformthe [45(cid:133)47] matureandstable9ScytoplasmicAHRcorecomplex Arylhydrocarbonreceptor AssociationofXAP2intotheAHRcomplexwithHSP90andp23stabilizes [12,45(cid:133)48,53,59] AHRandorsfavasmiccytoplretentionofAHRtolimitAHR-dependent transcription.,erselyvConstabilizationofAHRalsoappearstoincrease theligandvitysensitiofAHR,wingallogreatertranscriptionofAHR- dependentgettargenes Glucocorticoidreceptor BindingofXAP2toGRrequiresHSP90andservestolimitGRvenessresponsi [84] toglucocorticoidligands,possiblythroughstabilizationandoplasmiccyt retention Peroxisome-proliferator SimilartoGR,theassociationofXAP2withARPP a andHSP90limits [83] atedvactireceptoralpha ationvtransactiofARPP a targetgenessionexpre Thyroidhormonereceptor b 1 XAP2enhancesthetranscriptionalvityactiofTR b 1utbnotTR b 2throughan [86,87] (TR b 1) unidenti(cid:147)edmechanism Phosphodiesterase2A(PDE2A) InteractionwithXAPincreasesthephosphodiesterasevityactiofPDE2Aand [97,102] limitsthecAMP-dependenttranslocationandationvactiofAHR Phosphodiesterase4A5 AssociationwithXAP2diminishesthephosphodiesterasevityactiofPDE4A5 [98,102] (PDE4A5) andthusmayateveleintracellularvelsleofcAMP vinSurvi(BRIC5) BindingtoXAP2stabilizesvinsurvitoatevelethecellularantiapoptotic [88,102] threshold Epstein(cid:133)Barrvirus-encoded AssociationbetweenA-3EBNandXAP2in(cid:148)uencesligand-mediated [82] nuclearantigen-3 ationvactiofAHR AHR xcomple results in a marked retention of AHR in are not matched by their Ahr -null counterparts [75]. These the oplasmiccyt compartment, probably through disruption non-AHRXAP2escomplexandtheirbiologicalimplications of the association of AHR with the nuclear transport ma- are outlinedinableT3.1. .chinery Such retention obviously sequesters AHR away TheerydiscovofXAP2wastheresultofyeasttwo-hybrid fromthesiteofitsprimaryfunctionasatranscription,factor assays performed to identify proteins that interact with the that is, the nucleus. wHo these two opposing phenomena HBV X protein [76]. HBx is a promiscuous indirect tran- canbereconciledremainstobeestablished,anditsuggests scriptional atorvacti that appears to be required for viral that XAP2 regulation of AHR function is a highly ordered replication and thus contributes to the pathology of HBV and xcompleprocess. infection[77(cid:133)80].HBxisthoughttopromotecirrhosisofthe verli and hepatocarcinoma in -infectedHBV vidualsindi through multiple mechanisms [81]. Despite the yvelrelati 3.2.4 Insights from XAP2 Activity in Other wloexpression ofXAP2 inhepatocytes, studiesvealrethat Protein Complexes XAP2 can abolish HBx-mediated ationvtransacti [76] and Since the initial cloning of XAP2, its role in the cell has through this mechanism may provide some degree of pro- predominantlybeenstudiedandcharacterizedinthecontext tection against HBV replication and associated verli dys- ofitsassociationwithAHR;,verwehoanincreasingnumber function. XAP2 is also reported to interact with another ofadditionalinteractionsehavrecentlybeendemonstrated, virally encoded protein, Epstein(cid:133)Barr virus nuclear anti- whichsuggeststhatXAP2hasphysiologicalrolesindepen- gen-3A-3)(EBN[82].Althoughthisassociationisreported dentofAHR.Evidenceforsuchrolesisprovidedby-velde toin(cid:148)uenceligand-dependentationvactiofAHR,thefectef opmental expression studies, which illustrate that XAP2 of XAP2 on-mediatedEBVlymphocyte pathology has not expressionprecedesthatofAHRandthatXAP2expression been established. XAP2 has been demonstrated to be a exhibits a more ubiquitous tissue expression pro(cid:147)le than componentofnuclearreceptorxes,compleforexample,GR, AHR [74]. Furthermore, ablation of XAP2 in homozygous peroxisomeatedvproliferator-actireceptor a AR(PP a ),and Xap2 nullmiceresultsinembryoniclethalphenotypesthat thyroidhormonereceptors(TR b ).Theseinteractionsrequire 54 OLEROFONECHAPERTEINSOPRINAHRFUNCTION FIGURE3.3 Homologybetweentheco-chaperoneXAP2andtheimmunophilins.XAP2sharesa common domain structure with many of the FK506 binding immunophilin family of proteins consistingofanamino-terminalFKpeptidylprolylisomerasedomainthatdoesnottexhibiFK506 bindingorisomerasevityacticommontotheimmunophilinssuchasFKBP52.Thecarboxyl-terminal half of XAP2 encompasses 3 (cid:3) TPR protein(cid:133)protein interaction domains, the most C-terminal of whichfacilitatesitsinteractionwithAHRandHSP90. binding of XAP2 to HSP90 through the TPR domains and which then diminishes the stabilizing fect.ef,Clearlythese appeartolimitvenessresponsitoligandinthecaseofGRand interactionsimplicateXAP2asfactorthatmayimpacttumor ARPP[83,84].ItispossiblethatinthiscontextXAP2may wthgroandprogressionandthusrequiresfurthervestiga-in mimictheroleofFKBP51,whichiserexpressedovinwNe tion (Fig. 3.3). While no data are currently ailable,av the orldW primates and in part contributes to their verelati actionofXAP2indeterminingapoptoticsignalingmayehav vityinsensititoglucocorticoids[85].,verselyConTR b tran- particular ancevrele in the context of AHR. AHR is well scriptional vityacti is diminished in an isoform-speci(cid:147)c characterized as a mediator of xenobiotic-induced carcino- fashion wingfollo A-mediatedsiRN wnknockdo of XAP2, genesis through the induction of CYP1A1 and subsequent with TR b 1 being in(cid:148)uenced while TR b 2 vityacti is unaf- biotransformationofprocarcinogens(e.g.,benzo[ a ]pyrene) fected. This implies that XAP2 speci(cid:147)cally stabilizes intogenotoxicintermediates.ationvActiofAHRultimately TR b 1,thusfacilitatingthyroidhormone-mediatedtranscrip- leadstodissociationofXAP2fromtheAHRxcompleandthe tion[86, 87]. fateofliberatedXAP2remainstobeestablished.Itmaybe In addition to in(cid:148)uencing immediate ligand-induced speculatedthatonceXAP2isliberated,itmayassociatewith transcriptional responses, videncee exists to suggest that proteinssuchasRET51orvinsurviandthusmaycontribute XAP2mayalsofunctionasadeterminateofcellfatethrough wardtothetumorigenicpropertiesofcarcinogenicxenobio- nontranscriptionalmodesofaction.Forexample,XAP2has tics that bind to the AHR. beendemonstratedtointeractwithvinsurvi( BRIC5 )through ,RecentlyageneticlinkagehasassociatedXAP2withthe itsTPR-containingC-terminalend[88,89].Asamemberof occurrence of familial and sporadic isolated pituitary ade- the inhibitor of the apoptosis family of proteins, vinsurvi nomawithXAP2mutations(49mutationscharacterizedto serves as a alvsurvi factor by inhibiting the proapoptotic date)foundin15(cid:133)40%ofcases[90(cid:133)93].Inthemajorityof vityactiofedcleavcaspases3and7.,Interestinglyvinsurviis cases, the mutations resulted in truncated or nonsense ex- highlyessedexprintumorsandthuslikelycontributestothe pressionofXAP2,withthelossoftheTPRdomain.Sincethe malignant phenotype. The interaction between XAP2 and TPR domain is the primary site of protein(cid:133)protein interac- vinsurviappearstoenhancethestabilityofvin,survirender- tionforXAP2,itislikelythatthesefunctionsofXAP2are ingcellslessvesensititoprogrammedcelldeath.,Recently lostordiminished.Itisunclearbywhichmechanismtheloss XAP2hasbeenwnshotoassociate invivo withthetyrosine of XAP2 function contributes to the speci(cid:147)c velopdmenet receptorkinaseRET51[89].RET51servesasthereceptorfor and progression of pituitary adenomas. In vitro studies glialcellvedline-derineurotrophicfamilyofwthgrofactors examiningthefectefofpathogenicXAP2mutationsvealre and has the capacity to transduce proapoptotic stimuli, no disruption in the context of RET51 binding. Similar resulting in cell death. The XAP2/RET51 association has studies examining the vsuinrvi protein ehav yet to be per- been suggested to promote apoptosis by a mechanism in- formed; therefore, the role of mutated XAP2 and its inter- vingvol the sequestration of XAP2 ayaw from vin,survi action with these cell fate factors with regard to pituitary NCOMPOSITIOOFTHEDEDUNLIGANAHRCORECOMPLEX 55 pathogenesisremaintobeestablished.Otherpotentialme- physiological signi(cid:147)cance of its interaction with XAP2. chanismsmayvolveintheendocrinenatureofthepituitary ,Intriguinglyationveleofintracellular,cAMPaswouldoccur gland. As highlighted ,viopusrely XAP2 can speci(cid:147)cally through inhibition of PDE4A5, such as with rolipram or enhance TR b 1-mediated gene expression, with increased reduced phosphodiesterase ,vityactihas been wnsho to at- expressionofhypothalamicTSHbeingtheprimeexample. tenuate in(cid:148)ammatory responses and is currently an veacti Also,thefectefofXAP2ondiminishedGRsenvitsityimay areaofresearch.Itcouldbespeculatedthatsomeoftheanti- together promote an endocrine disrupting fectef on the in(cid:148)ammatory fectsef may arise from liberation of XAP2 hypothalamic(cid:133)pituitary(cid:133)adrenalaxis,thusprovidingapro- from the AHR xcomple (which has also been wnsho to tumorigenichormonalvironmenent.,ClearlyXAP2mustbe exhibitanti-in(cid:148)ammatoryproperties)wingfolloligandacti- providing a tumor suppressor function, which is lost upon ation.v This free XAP2 might then subsequently bind to mutation,and thusadditionalstudiesarerequired to delin- PDE4A5 and attenuate its catalytic,vityactithus atingvele eate the mechanisms by which wild-type XAP2 ertsex its .cAMP,verweHo such a mechanism remains to be estab- suppressor.vaitctiy,Currentlynolinkageanalysishasbeen lished.Thesecondisotypeofphosphodiesterasedemonstrat- performed or detected with other nonpituitary tumors, but ed to bind XAP2 is PDEA2. Similar to PDE4A5, PDEA2 vengithealencvpereofhepatitisB-associatedhepaticcancer binding is mediated through the XAP2 TPR domain; w-ho andtheinteractionbetweenHBxandXAP2,itislikelythat ,vereunlikePDE4A5,PDEA2utilizesaferentdifbindingsite this interaction hassome impacton hepatic tumorigenesis. thatesvolvintheGAF-Bcyclicnucleotidebindingsite[97]. ,UnfortunatelyXAP2nullanimalmodelsarenotailablaveto ,InterestinglydespiteutilizingtheGAF-Bdomain,bindingof exploretheroleofXAP2incarcinogenesisduetoembryonic XAP2 does not attenuate the catalytic vityacti of PDEA2, lethality [75].,verweHothe velopmedent of tissue-speci(cid:147)c which is in contrast to PDE4A5, suggesting that XAP2 conditionalknockout,mutant,orhypomorphicmodelsmay interaction serves two distinct roles with gardre to these proveinformveati[94]. phosphodiesteraseisotypes.UnlikethePDE4A5interaction, AnothermechanismbywhichXAP2maymodulatecel- the binding of XAP2 to PDEA2 has been demonstrated to lular function is through its association with phosphodies- ertexafunctionalfectefonAHRsignaling[97].Anumberof terases, enzymes that degrade the second messenger cyclic groupsehavhighlightedthephenomenonofligand-indepen- nucleotidescAMPand.cGMPPhosphodiesterasesareubiq- dent AHR ationvacti wingfollo exposure to cAMP or the uitouslyexpressedandtherearenumeroussubtypesencoded adenylatecyclaseatorvactiforskolin,oftenwithcon(cid:148)icting by ferentdif genes with ferentdif modes of regulation and ationsobserv that are attributed to species, cell, or tissue nucleotide speci(cid:147)city[95,96].wo-hybridTassays andsub- ferencesdif[99(cid:133)101].DataindicatethatcAMPoritsanalogs sequentGSTwnpull-dostudiesehavidenti(cid:147)edandalidatedv promotenucleartranslocationofAHRandsubsequenttran- twophosphodiesterases,,namelyPDE2AandPDE4A5,that scriptionofAHRtargetgenes;,verwehoforcedexpressionof interactwithXAP2[97,98].BothPDE2AandPDE4A5were PDEA2 through transient transfection results in increased wnshotoassociatewithXAP2throughtheC-terminalTPR oplasmiccyt retention of AHR under basal, TCDD-, and domain.,Interestinglydespitesharingthesamebindingsite cAMP-inducedstates[97,99,102].Undertheseconditions, onXAP2,thedomainsontheverespectiphosphodiesterase the reduction in AHR vityacti is attributed to increased isotypesthatfacilitatetheinteractionareferent,difsuggest- binding of XAP2 to PDEA2. Such data may indicate that ingtwoindependentmodesofXAP2binding[97,98].Inthe PDEA2 forms a xcomple with AHR mediated through a caseofPDE4A5,theinteractionishighlyspeci(cid:147)cforXAP2 mutual interaction with XAP2, an interaction that may be since there is no videncee for any association with the further stabilized through the ementvolvin of HSP90. The immunophilins, for example, FKBP52 [98]. ,Similarly presenceofPDEA2asacomponentoftheAHRxcomplehas amongthe PDE4 isotypes,PDE4A5appearstobetheonly beenspeculatedtodiminishthelocalcAMPconcentrationin onethatxescomplewithXAP2.Thefunctionalancesigni(cid:147)c thevicinityofAHR,thusdecreasingitscapacitytoundergo oftheXAP2(cid:133)PDE4A5interactionhasyettobeelucidatedin nucleartranslocation.Suchamechanismmayprovidescope thecontextofAHRsignaling,butformationofthiscomplex forthesubtleregulationofAHRvityactiorwallocontextual hasprofoundfectsefonPDE4A5function.Thephosphodi- ationvacti or inhibition of AHR when multiple signaling esterasecatalyticvityactiofPDE4A5isgreatlydiminished stimuli areertedex(Fig. 3.4). by60%anditsvitysensititothephosphodiesteraseinhibitor Thus,inconclusion,XAP2appearstoehavedvolvetobea rolipramisenhancedwhenboundtoXAP2[98].Inaddition, satilever pleiotropic factor with the capacity to modulate XAP2 attenuates the capacity of protein kinase A to phos- severdibiologicalvitiesactithroughnumerousproteininter- phorylate PDE4A5. Data are limited regarding the physio- actions. Evidence suggests that some of these interactions logicalroleofPDE4A5,butithasbeenimplicatedinsperm may in(cid:148)uence the vityacti and biological functions of the ,motility apoptosis, in(cid:148)ammation, and adipocyte metabo- mostwidelyexaminedXAP2bindingpartnertheAHR.Itis lism. venGi the lack of information at this time regarding likelythatfurthervestigationinwillidentifyadditionalelnov the role of PDE4A5, it is (cid:147)cultdif to ascertain the precise XAP2-interactingproteinsandprovidealuablevinformation