REVIEW AIP and its interacting partners - Journal of Endocrinology

19 Pages·2011·0.4 MB·English

137
REVIEW
AIP and its interacting partners
Giampaolo Trivellin1,2 and Ma´rta Korbonits1
1DepartmentofEndocrinology,Bart’sandtheLondonSchoolofMedicine,QueenMaryUniversityofLondon,LondonEC1M6BQ,UK
2EndocrinologyDivision,DepartmentofMedicalandSurgicalSciences,ViaOspedale,105,35128Padova,Italy
(CorrespondenceshouldbeaddressedtoMKorbonits;Email:[email protected])
Abstract
Germline mutations in the aryl hydrocarbon receptor- (TPR domains) that mediate the interaction of AIP with
interacting protein gene (AIP) predispose to young-onset most of its partners. In this review, we summarise the
pituitarytumours,most oftentoGH- or prolactin-secreting known interactions of AIP described so far. The
adenomas, and most of these patients belong to familial identification of AIP partners and the understanding of
isolated pituitary adenoma families. The molecular pathway howAIPinteractswiththeseproteinsmighthelptoexplain
initiated by the loss-of-function AIP mutations leading to the specific phenotype of the families with heterozygous
pituitary tumour formation is unknown. AIP, a co-chaper- AIP mutations, to gain deeper insight into the pathological
one of heat-shock protein 90 and various nuclear receptors, process of pituitary tumour formation and to identify novel
belongs to the family of tetratricopeptide repeat (TPR)- drug targets.
containing proteins. It has three antiparallel a-helix motifs JournalofEndocrinology(2011)210,137–155
Introduction feature in patients with AIP mutation (Chahal et al. 2011).
Occasionally,patientswithyoung-onsetacromegalyorother
Familial pituitary adenomas can occur in three diseases: childhood-onsetpituitaryadenomascarrygermlinemutation
multiple endocrine neoplasia type 1 (MEN1, MIM# in the AIP gene without apparent family history (Cazabat
131100), the rare Carney complex (CNC, MIM# 160980) et al. 2007, Georgitsi et al. 2008, Daly et al. 2010, Stratakis
andtherecentlydescribedfamilialisolatedpituitaryadenoma et al. 2010). The penetrance of the disease is low, probably
(FIPA, MIM# 102200). Inthe first two syndromes multiple around 30%, and there is a male preponderance (Cain et al.
endocrine organs are involved, whereas in FIPA tumours 2010,Dalyetal.2010).Three-quartersofAIPmutationslead
occuronlyinthepituitarygland(Chahaletal.2010).Alocus to a truncated protein, and some of the missense mutations
has previously been identified in a phenotypically relatively have also shown to result in loss of function of the protein
well-definedsubgroupofFIPAfamilies,butthegenecausing suggesting, together with loss of heterozygosity data, that
thedisease,namelythearylhydrocarbonreceptor-interacting AIP functions as a tumour suppressor gene in the pituitary
protein gene (AIP, MIM# 605555), had not been identified gland(Soaresetal.2005,Vierimaaetal.2006,Leontiouetal.
until 2006 (Benlian et al. 1995, Gadelha et al. 2000, Soares 2008,Igrejaetal.2010).
et al. 2005, Vierimaa et al. 2006). About 30% of all FIPA At first, AIP seems to be an unusual gene causing
families and 50% of acromegaly families have a mutation in pituitary adenoma as it was previously only known as a
the AIP gene (Chahal et al. 2010). Families with AIP co-chaperone of nuclear receptors or viral proteins
mutations have a characteristic phenotype: childhood- or (Kuzhandaivelu et al. 1996, Carver & Bradfield 1997, Ma
young-onset disease (the mean age at diagnosis is around 20 & Whitlock 1997, Meyer et al. 1998, Kashuba et al. 2000).
years), primarily GH- or prolactin-secreting adenomas (the AIP, also known as X-associated protein-2 (XAP2;
majority of the patients have GH- or mixed GH- and Kuzhandaivelu et al. 1996), Ah receptor-activated 9
prolactin-secretingadenomas,aminorityhaveprolactinomas, (ARA9; Carver & Bradfield 1997) or FK506-binding
whereas other pituitary adenoma types are rarely observed), protein 37 (FKBP37; Blatch et al. 2006), is a 37kDa
andlargeandinvasivepituitarytumoursthatdonotrespond cytoplasmic protein. Structurally, it shares a significant
welltosomatostatinanaloguetreatment(Leontiouetal.2008, degree of homology with immunophilins, such as FKBP52
Daly et al. 2010). Pituitary apoplexy can be a presenting (52kDa FK506-binding protein), as it has a peptidyl-prolyl
JournalofEndocrinology(2011)210,137–155 DOI:10.1530/JOE-11-0054
0022–0795/11/0210–137 q2011SocietyforEndocrinology PrintedinGreatBritain Onlineversionviahttp://www.endocrinology-journals.org
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138 GTRIVELLINANDMKORBONITS AIPinteractingpartners
cis–trans isomerase (PPIase)-like domain. Immunophilins AIP-interacting proteins
are a huge family of ubiquitous and conserved proteins,
which possess PPIase domains that bind immunosuppressant Viralproteins
drugs of the FK506 or of the cyclosporin A groups (Galat
HepatitisBvirusXproteinAIPwasoriginallydescribed
& Metcalfe 1995). However, AIP does not function as an
as a protein associated with the X protein of the hepatitis B
immunophilin. AIP lacks affinity for the immunosuppres-
virus(HBV;Kuzhandaiveluetal.1996),asmallhumanDNA
sant drugs FK506 and rapamycin and the PPIase-like
virusthatcausesacuteandchronichepatitis.Amongthefew
domain displays no enzymatic activity (Carver et al. 1998,
genes contained in the genome of HBV, there is an open
Laenger et al. 2009), so AIP cannot be considered a true
reading frame that encodes a 154 amino acid regulatory
immunophilin. These data are consistent with a weak
protein,termedXprotein.Thisprotein,whichdoesnothave
homologybetweenthePPIasedomainsofAIPandFKBP12
a human homologue, activates the transcription of a wide
(onlyfiveofthe14aminoacidsoftheFK506-bindingdomain
variety of different genes through interaction with cellular
of FKBP12 are conserved in AIP (Carver et al. 1998)) and
factors (Koike 2009). In order to identify new proteins that
explainthedifferentbiochemicalpropertiesofAIP.
may interact with X, the authors used the yeast two-hybrid
AIP belongs to the family of tetratricopeptide repeat
(Y2H)method.AmongtheseveralpotentialcDNAscoding
(TPR) domain-containing proteins, such as the aryl
the protein X binding protein, six were found to be
hydrocarbon receptor-interacting protein-like 1 (AIPL1),
overlapping clones of a full-length cDNA encoding the
protein phosphatase 5 (PP5), FK506-binding protein 51
same gene. The gene was named XAP2 because it was the
(FKBP51), FKBP52, cyclophilin 40 (Cyp40), carboxyl
secondproteinfoundtointeractwiththeHBVXproteinby
terminus of Hsc70-interacting protein (CHIP), and heat-
thistechnique.Thefull-lengthAIPcDNAwassubsequently
shock protein 70 (Hsp70)/Hsp90 organising protein (Hop;
isolated and in vitro translated in a rabbit reticulocyte lysate.
D’Andrea & Regan 2003), and has three TPR motifs and a
Thetranslatedproductcorrespondedperfectlywiththenative
finala-7helixattheC-terminus(Fig.1).TPRdomainsare
AIPfromHeLacellsandhadanapparentmolecular massof
highly degenerate consensus sequences of 34 amino acids,
36kDa.AIPRNAexpressionwasevaluatedanddetectedin
often arranged in tandem repeats, formed by two a-helices
severaldifferenttissuesandcelllines,butverylowlevelswere
forminganantiparallelamphipathic(havingbothhydrophilic
found in the liver. The ubiquitous expression of AIP was
and lipophilic properties) structure that mediates intra- and
subsequently confirmed in human and murine tissues at the
inter-molecular interactions in many proteins (Goebl &
mRNA and protein level (Kuzhandaivelu et al. 1996, Ma &
Yanagida1991).
Whitlock1997,Carveretal.1998,Meyeretal.1998,2000,
The AIP protein sequence is evolutionarily conserved
Yanoetal.2003).
among species. The protein sequence of human AIP is 100,
The X protein–AIP interaction was also demonstrated
94 and 93% identical to chimpanzee, mouse and rat AIP
to occur in vitro by testing the ability of a glutathione
respectively (Supplementary Table 1, see section on supple-
mentary data given at the end of this article). Furthermore, S-transferase(GST)–Xfusionproteintobindtoa35S-labelled
AIPislocatedonaconservedsyntheticblockinthehuman, AIP. UsingdifferentXmutants,theinteractionwithAIPwas
mouseandratgenome(Fig.2).ThefactthatAIPisahighly then shown to be mediated by X protein residues 13–26, a
conserved protein could be expected for two reasons: first, regionhighlyconservedamongallmammalianhepadnaviruses.
because AIP is associated with a human disease, and several Further evidence regarding this interaction shows similar
studieshavefoundthatgenescausinghumandiseasearemore cytoplasmicdistributionsofXproteinandAIPwithimmuno-
conserved than non-disease genes (Lovell et al. 2009), and cytochemistryinXprotein-transfectedmammaliancells.
secondly,becauseAIPhasbeendemonstratedtobeessential Overexpressing AIP resulted in inhibited X protein
in cardiac development and in maintaining productive transcriptional activity, suggesting that AIP is an important
erythropoiesis in mice (Lin et al. 2007, Kang et al. 2011) negativeregulatoroftheXproteinandthattheir interaction
and previous studies in mammals and other eukaryotes mayplayaroleinHBVpathology.
showed that essential genes are usually located in highly
conservedgenomicregions(Lovelletal.2009). EBNA-3 EBV-immortalised lymphoblastoid cell lines
AsexpectedfromthepresenceofTPRmotifsintheAIP express, among others, six nuclear antigens (EBNA 1–6)
protein, several proteins have been identified which interact andthreelatentmembraneproteins(LMP1,2a,2b),whose
with AIP. The identification of these molecules and the concerted action is essential for the immortalisation and
understanding of how AIP interacts with them could give transformation of B-cells (Tomkinson et al. 1993). As the
us an insight into the pathological process of pituitary identification of cellular proteins that can interact with the
tumour formation and may lead to new therapeutic targets. transformation associated EBNAs was not yet complete,
The main focus of this review is thus to summarise the Kashubaetal.(2000)searchedforproteinsthatcanbindtothe
AIP-interacting partners described so far. The functional transcriptionalregulatorEBNA-3,whichagaindoesnothave
studies available about specific AIP amino acid mutations a human homologue, in a Y2H system. Among the several
are also reviewed. clones identified, one corresponded to the AIP protein.
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AIPinteractingpartners GTRIVELLINANDMKORBONITS 139
G272D/
G272E
Y268A R304Q
A R271W E293G
C238Y I257V F288A
R16H V49M K103R N236A K241E K266A A284T A299V
PPIase-like TPR1 TPR2 TPR3 α-7
31 121 179 212 231 264 298 330
B
PPIase-like
TPR1 TPR2
TPR3 α-7
Figure1 (A)SchematicstructureoftheAIPprotein.Structurally,AIPissimilartoimmunophilins.AIPcontainsaPPIase-likedomainatthe
N-terminus,whichshowsaweakidentitytothelowmolecularweightFKBP12(12kDaFK506-bindingprotein)(Carver&Bradfield1997),
butdoesnotshowimmunophilinactivity(Carveretal.1998,Laengeretal.2009).TheC-terminalpartofthemoleculecontainsthreeTPR
domainsthatareconserved34aminostructuresconsistingoftwoantiparallelahelices.Thereisaterminala-7helixthatiscrucialfor
protein–proteininteraction.Numbersunderneaththeproteinstructurerepresentaminoacids.Boxeswithdifferentshapesrepresent
differentdomains.Thelocationofthemissensemutationsinvestigatedinfunctionalbindingassaysismarked.PPIase-like,peptidyl-prolyl
cis–transisomerase-likedomain;TPR,tetratricopeptiderepeatdomain.(B)HypotheticalstructureofAIPbasedonthecrystalstructureof
therelatedproteinFKBP51(Igrejaetal.2010).ThePPIase-likedomain,thethreeTPRmotifswiththreepairsofanti-parallela-helicesand
thefinalextendeda-helix,a-7,arehighlighted.Theaminoacidssubjectedtosite-directedmutagenesisanalysestostudytheirroleinthe
AhRcomplexareshowninred(Bell&Poland2000,Meyeretal.2000,Laengeretal.2009).
Subsequently,thisinteractionwasalsoconfirmedinvitrousing AIP–AhR–Hsp90 complex
aGSTpull-downassay.
More or less parallel to the discovery of the binding with
Concordant to that shown for the aryl hydrocarbon
HBV X protein (Kuzhandaivelu et al. 1996), AIP was
receptor (AhR (MIM# 600253); see AIP–AhR–Hsp90
independentlyidentifiedbythreelaboratoriestoalsointeract
section), AIP was also found to translocate to the nucleus
withtwootherproteins:theAhR(Carver&Bradfield1997,
on expression of EBNA-3. The authors hypothesised that
Ma & Whitlock 1997, Meyer et al. 1998) and the hsp90
as AIP can bind to the transforming proteins of two
(Carver et al. 1998, Meyer & Perdew 1999, Bell & Poland
evolutionarily distant viruses and also to the AhR, a
phylogenetically ancient protein conserved in vertebrates 2000, Kazlauskas et al. 2002, Yano et al. 2003, Laenger et al.
and invertebrates (Hahn 2002), the AhR signal transduction 2009,Schulkeetal.2010).
pathway could be involved in virus-induced cell transfor-
mation.Thishypothesiswasfurthersupportedbyafollowing AIP–AhR The AhR, a basic helix–loop–helix protein
demonstrationfromthesamegroupthatbesidesAIP,EBNA- of the Per–ARNT–Sim (PAS) family of transcriptional
3canalsodirectlyinteractwiththeAhR,withAIPenhancing regulators, is a cytoplasmic transcription factor that can be
the stability of the complex. As a result of this association, activated byawidevarietyof structurally diverse exogenous
anenhancedtranscriptionofAhR-responsivegeneshasbeen ligands, the prototype of which is the 2,3,7,8-tetrachloro-
observed(Kashubaetal.2006). dibenzo-p-dioxin (TCDD), as well as by some endogenous
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140 GTRIVELLINANDMKORBONITS AIPinteractingpartners
Rat Mouse Human Human Human Mouse Mouse Mouse
chr 1q2 chr 19qA chr 11q13 position Gene chromosome gene position
206730K 4145K GPR152 11:67,195,935-67,202,879 (+) RPS6KB2 19 Rps6kb2 19:4,156,977-4,163,245 (–)
Gpr152
11:67,202,981-67,205,153 (–) PTPRCAP 19 Ptprcap 19:4,154,646-4,156,710 (+)
LOC688884 4140K CABP4 11:67,205,518-67,211,263 (–) CORO1B 19 Coro1b 19:4,148,663-4,154,035 (+)
206725K
Cabp4
11:67,218,772-67,220,200 (–) GPR152 19 Gpr152 19:4,139,799-4,145,741 (+)
4135K
Cabp4_pr.. 11:67,222,818-67,226,691 (+) CABP4 19 Cabp4 19:4,135,423-4,139,609 (–)
206720K
TMEM134 11:67,231,819-67,236,731 (–) TMEM134 19 Tmem134 19:4,125,960-4,132,307 (+)
4130K
Tmem134 11:67,250,505-67,258,579 (+) AIP 19 Aip 19:4,114,446-4,125,827 (–)
206715K
11:67,259,239-67,272,843 (–) PITPNM1 19 Pitpnm1 19:4,100,622-4,113,961 (+)
RGD13058.. 4125K
11:67,273,968-67,276,102 (–) CDK2AP2 19 Cdk2ap2 19:4,097,351-4,099,017 (+)
206710K
11:67,286,418-67,290,899 (–) CABP2 19 Cabp2 19:4,083,490-4,087,340 (+)
4120K Aip
11:67,374,323-67,380,012 (+) NDUFV1 19 Ndufv1 19:4,007,505-4,012,725 (–)
206705K
1 194115K AIP
Aip
206700K
4110K
206695K Pitpnm1
4105K PITPNM1
Pitpnm1
206690K
4100K
Cdk2ap2
206685K CDK2AP2
4095K
LOC688405
Figure2 Human–mouse–ratsyntenymaparoundtheAIPlocus(highlightedinred)(http://www.ncbi.nlm.nih.gov/projects/homology/
maps/human/chr11/).
compounds, such as the cAMP (Denison & Nagy 2003, the AhR (Carver & Bradfield 1997, Ma & Whitlock 1997,
Oesch-Bartlomowiczetal.2005,Nguyen&Bradfield2008). Meyer et al. 1998, 2000, LaPres et al. 2000, Nukaya et al.
Ligand-freeAhRinthecytoplasmbindstotwomoleculesof 2010), whereas others described an inhibitory function
hsp90 (Perdew 1988) and the co-chaperone proteins p23 (Hollingsheadetal.2004,Pollenz&Dougherty2005,Pollenz
(Nair et al. 1996) and AIP (Carver & Bradfield 1997, Ma & etal.2006).Thisvariabilityisduetoseveralfactors,including
Whitlock 1997, Meyer et al. 1998). The interaction with species- and tissue-specific differences. Apart from these
hsp90 shapes the AhR’s ligand-binding domain into a state conflicting results, AIP was repeatedly shown to protect the
competentforligandbinding,anditalsonegativelyregulates AhR from ubiquitin-dependent degradation through the
AhRuntilligandbindingoccurs(Beischlagetal.2008).p23 proteasome(Kazlauskasetal.2000,Morales&Perdew2007).
is part of the AhR complex through interaction with hsp90 Consistentwiththat,lowlevelsorlossofAIPcorrelateswith
(Nair et al. 1996) and its presence is thought to stabilise the lowexpressionofAhR(Jaffrain-Reaetal.2009).
complex (Kazlauskas et al. 1999) and to favour its nuclear After ligand binding, AhR undergoes a conformational
import (Kazlauskas et al. 2001). These functions are in line changethatexposesanuclearlocalisationsequence,resulting
with the modulating role that p23 has been shown to exert in translocation of the complex into the nucleus, where it
in different steroid hormone receptors (Smith et al. 1995, forms a heterodimer with the aryl hydrocarbon receptor
Dittmaretal.1997,Freemanetal.2000).Thepresenceofp23 nucleartranslocator1(ARNT,alsoknownasHIF1b,MIM#
intheAhRcomplexseems,however,nottobeessentialfor 126110),whichleadstotheactivationofAhR-sensitivegenes.
theAhRphysiology(Cox&Miller2004,Flavenyetal.2009). Inmice,ligandbindingleadstothedissociationofAIPfrom
Using the Y2H method with AhR as the bait, a human thecomplexasitentersthenucleus(Ma&Whitlock1997),
cDNA(termedatthetimeARA9;Carver&Bradfield1997) whereas in humans the association is maintained in the
and a murine cDNA (termed AIP) were described (Ma & nucleus(Carver&Bradfield1997,Ramadossetal.2004).
Whitlock1997),bothshowntobeidenticaltoXAP2.Atthe In addition to its well-established role as a transcription
sametime,sinceanuncharacterisedproteinofabout43kDa factor, the ligand-activated AhR has also been shown to
was found to be part of the AhR–hsp90 complex (Chen & modulatethefunctionsofothertranscriptionfactors,suchas
Perdew1994),athirdgroupdecidedtoidentifythisprotein. the oestrogen receptor (ERa and ERb) and the androgen
After purification from simian COS-1 cells, the molecular receptor (AR). The crosstalk of AhR with ER and AR
mass was reassigned from 43 to 38kDa and the protein explicates via a direct association in the nucleus, which
sequence was found to be 98% identical to human AIP modulates oestrogen/androgen signalling both positively
(Meyeretal.1998). and negatively depending on the cellular context (Ohtake
There is considerable controversy regarding the effect of et al. 2009, Pongratz et al. 2009). Furthermore, a novel and
AIPon AhR function. Some studies reported that AIP can unexpectedroleforAhRasaligand-dependentE3ubiquitin
enhance the transcriptional activity and expression levels of ligase has also recently been described: the ligand-bound
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AIPinteractingpartners GTRIVELLINANDMKORBONITS 141
AhR promotes the ubiquitination and proteasomal ARNTwas thought to be a good candidate because it
degradation of ER and AR through the assembly of a belongstothesamefamilyoftranscriptionfactorsofAhRand
ubiquitin ligase complex, referred to as CUL4BAhR. AIP, both share a similar modular structure. However, the results
however, seemed unlikely to influence the CUL4BAhR- fromthreedifferentstudiesdemonstratedthatAIPisexcluded
mediatedERdegradation(Ohtakeetal.2007). from the AhR–ARNT heterocomplex in vitro and in vivo
(Carver & Bradfield 1997, Ma & Whitlock 1997, Meyer &
AIP–Hsp90 Hsp90 is a highly abundant molecular Perdew1999).ThesefindingsagreewiththeAhRmapping
chaperone, which associates as a dimer with a set of highly data because AIP and ARNT have been shown to contact,
different client proteins. Hsp90 is required to maintain at least in part, the same or an adjacent binding site on the
signalling proteins in an active conformation that can be AhR(Meyer&Perdew1999).EvenifAIPdoesnotinteract
rapidly triggered by ligands. Hsp90 functions as the core with ARNT, two recent studies showed that the expression
component of a dynamic set of multiprotein complexes, of ARNT protein is significantly reduced in AIP-mutated
involving a set of co-chaperones. Structurally, hsp90 can be pituitary tumours, suggesting that loss of AIP leads to
divided into five domains: a highly conserved N-terminal an imbalance in the AhR–ARNT complex formation
domain involved in nucleotide and drug binding, a charged (Heliovaaraetal.2009,Raitilaetal.2010).
domain, a middle domain with ATPase activity involved in
p23 was then demonstrated to contact AIP in co-immu-
client protein binding, a second charged domain and a
noprecipitation (co-IP) experiments (Hollingshead et al.
C-terminal domain involved in dimerisation and binding of
2004), but only indirectly via hsp90, as previously demon-
TPR-containing proteins (mediated by a conserved EEVD
stratedforAhR(Nairetal.1996).AIPwasalsoshowntobe
motif)(Pearl&Prodromou2006).
able to displace p23 from the AhR complex, an effect only
ThedirectbutmoderateassociationofAIPwithhsp90has
specific for the AhR complex and not for hsp90 alone
been demonstrated in different studies (Carver et al. 1998,
(Hollingsheadetal.2004).
Meyer&Perdew1999,Bell&Poland2000,Kazlauskasetal.
2002, Yano et al. 2003, Laenger et al. 2009, Schulke et al.
AIP self-association An interesting finding was the
2010).Hyperacetylationofhsp90wasfoundtoleadtotheloss
evidence that AIP can exist in multimeric complexes of at
of complex formation with AhR, p23 and AIP (Kekatpure
least two molecules even without requiring AhR or hsp90
etal.2009).Discordantfindingsaroseabouttheroleexerted
(Hollingsheadetal.2004).Theself-associationofAIPisvery
by hsp90 in assisting the AIP–AhR interaction. A study
likely mediated by the TPR domain, as demonstrated for
demonstrated that AhR,in order to bind AIP, needs to fold
otherTPR-containingproteins(Dasetal.1998,Tayloretal.
into the mature ligand-binding conformation with the help
2001, Nyarko et al. 2007). This suggests that AIP can
of hsp90 (Bell & Poland 2000). This requirement of hsp90
homodimerise without the association of other auxiliary
was instead proved to be not essential in another report
proteins or at least others than AhR or hsp90 and also that
(Meyer&Perdew1999).
morethanonemoleculeofAIPcouldbepresentintheAhR
complex. However, two previous studies, using different
AIP–Hsc70 In the absence of AhR, AIP was shown to
stoichiometric approaches to examine the AhR complex
interact – with higher affinity – with another heat-shock
subunits composition, showed a AhR:hsp90:AIP ratio of
protein,theheat-shock cognate70(hsc70,MIM#600816),
1:2:1 (Chen & Perdew 1994, Petrulis et al. 2000). If these
rather than to hsp90 (Yano et al. 2003). Hsc70 is a
ratios are correct, the authors suggested that the multimeric
constitutively expressed co-chaperone protein that is
complexes of AIP act as a reservoir able to regulate the
involved, as hsp90, in protein folding and in mitochondrial
amountofavailablemonomericAIPthatcanbeincludedinto
protein import (discussed later in Translocase of the outer
membrane of mitochondria 20 (TOMM20) and mito- theAhRcomplex(Hollingsheadetal.2004)ormaybeinthe
chondrial preproteins section) (Young et al. 2003), but it othercomplexeswithwhichAIPhasbeenshowntointeract.
also functions as an ATPase in the disassembly of clathrin- Furthermore, the TPR-mediated self-association of AIP
coated vesicles during transport of membrane components might be a mechanism to specifically regulate its biological
through the cell (Alberts et al. 2002). Hsc70 is a member functions, as reported for the TPR-containing proteins
of the hsp70 family. Although human hsc70 shares 85% PP5 and Sgt1 (Yang et al. 2005, Nyarko et al. 2007). For
sequence identity with human hsp70, they play different example,thephosphataseactivityofPP5issuppressedbyan
cellularfunctions(Gething&Sambrook1992,Goldfarbetal. autoinhibitedconformationmaintainedbytheTPRdomain–
2006). Consistent with this, AIP was found to be unable catalyticdomaininteraction(Yangetal.2005).
tobindhsp70(Schulkeetal.2010).
Domains mediating AIP–AhR–Hsp90 interaction
AIP interactions with other proteins of the AhR and functional studies Different studies contributed to
signalling pathway Apart from AhR and hsp90, it was definethedomainsinvolvedinAIP–AhR–Hsp90interaction
also investigated whether AIP binds to two other proteins andalso,morespecifically,theAIPresiduesessentialforAhR
involvedintheAhRpathway,ARNTandp23. andhsp90binding.
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142 GTRIVELLINANDMKORBONITS AIPinteractingpartners
MappingexperimentsusingAIPdeletionmutantsshowed evenifitdidnotloosetheabilitytobindhsp90,wasexpressed
that the C-terminal half of AIP (residues 154–330), which at lower levels compared with the wild type (WT) protein,
contains the three TPR motifs, was necessary for binding maybe as a result of a higher turnover in cells (Meyer &
boththeAhRandthehsp90(Fig.3;Carveretal.1998,Meyer Perdew1999).
&Perdew1999,Kazlauskasetal.2000).Anindispensablerole In the reciprocal mapping analyses, the boundaries of
in mediating AhR binding was shown for the a-helical theAhRproteinsegmentinteractingwithAIPweredefined
C-terminus (a-7) of murine AIP: deletion of the last five to be approximately between amino acids 380 and 419,
amino acids abolishes almost completely AhR binding, which encompass the C-terminal portion of the PAS
without affecting AIP–hsp90 interaction (Bell & Poland domain (PAS-B; Carver et al. 1998, Meyer & Perdew 1999,
2000). However, another study presented evidences Kazlauskasetal.2000).Thisinteractionseemstobemediated
suggesting that the human final a-7 helix is more likely to bynonpolarorhydrophobicaminoacids(Hollingsheadetal.
bind hsp90 than AhR (Kazlauskas et al. 2002). These 2004). It was also established that AIP, like other immuno-
contrasting results might be explained by species-specific philins found in hsp90 complexes, binds to the C-terminal
differences.Thereare controversialresults regardingtherole segmentofhsp90(residues629–732),whereastheAhRbinds
playedbytheN-terminusofAIP.Somereportsdemonstrated to the middle region (residues 272–617) (Meyer & Perdew
that this region did not interact with either AhR or hsp90 1999, Bell & Poland 2000). Hsp90 was found to interact
(Carver et al. 1998,Meyer& Perdew1999, Kazlauskas et al. withtwospatiallydistinctmotifsoftheAhR,thePAS-Band
2000), whereas Kazlauskas et al. (2002) showed that the the bHLH domains (Antonsson et al. 1995). The domains
N-terminuscontainsanadditionalsiteofinteractionwiththe involved in the AIP–hsc70 interaction were not experi-
AhR complex. Moreover, the N-terminal part of AIP was mentallydetermined(Yanoetal.2003).
showntoconferstabilitytothecomplexandtobeessentialin Mutational analyses of some TPR domains indicate that
the regulation of the subcellular localisation of AhR theyperformessentialfunctions(Chenetal.1996,Blometal.
(Kazlauskasetal.2002).Takentogether,theseresultssuggest 2004)andtheinteractionwithhsp90throughTPRdomains
that despite the C-terminal region of AIP being capable of has been shown to be conserved in plants and animals
interacting alone with the AhR complex, this interaction is (Owens-Grilloetal.1996).Itis,therefore,verylikelythatthis
not functional. This hypothesis is further supported by the is a basic protein interaction critical to the function of AIP.
finding that an AIP mutant lacking the first 17 amino acids, Somestudiesaddressedthisissueanalysinghowmutationsin
Charged Charged
hsp90 N-terminus Middle domain C-terminus
domain domain
AhR bHLH PAS A PAS B Q-rich
AIP PPIase-like TPR1 TPR2 TPR3 α-7
Charged Charged
hsp90 N-terminus Middle domain C-terminus
domain domain
Figure3 LocationofkeyinteractingdomainsoftheAIP–AhR–Hsp90complex.Thesizeofdomainsisdrawntolinearscaleproportional
toaminoacidnumber.bHLH,basichelix–loop–helix;PAS,Per–ARNT–Simhomologydomain;Q,glutamin-richdomain.
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AIPinteractingpartners GTRIVELLINANDMKORBONITS 143
the TPR domain of AIP affect AhR and hsp90 binding, as The functional consequences of p.Y268A, p.G272D,
well as PDE4A5 and RET (rearranged during transfection; p.272E, p.A284Tand p.F288A AIP mutants on hsp90 and
describedinPDE4A5andRETsection). AhR binding were studied by Meyer et al. (2000) in the
Site-directedmutagenesisstudiesinthebindinggrooveof presence of both proteins or in the absence respectively, of
the TPR domain of PP5, an immunophilin co-chaperone one of them. In COS-1 cells (AhR-defective cells), each
protein present in the glucocorticoid receptor (GR, MIM# mutant was impaired in its ability to associate with hsp90,
138040) complex (Silverstein et al. 1997), confirmed the with only p.Y268A showing a residual level of binding,
prediction made from the previously discovered three- compared with the others in which no binding at all was
dimensional structure of the protein (Das et al. 1998) that observed. Vice versa, in the absence of hsp90, it was found
basicaminoacidresiduesinthisregionareimportantforhsp90 thatonlythep.Y268Aandthep.A284Tmutationswereable
binding(Russelletal.1999).Astheseresiduesareconservedin to bind to the AhR, whereas the others were not. In the
theTPRdomainsofotherhsp90-interactingproteins,suchas presenceoftheAhR–hsp90complex,itwasthenfoundthat
FKBP51,FKBP52andAIP,theyarelikelytobeoffunctional alltheAIPmutantscanbindAhR–hsp90complexexceptthe
significanceinordertomediateefficientinteractionswiththeir p.G272D and the p.G272E mutants. The following four
binding partners (Fig. 4). Based mainly on this assumption, conclusionscanbedrawnfromtheresultsofthisstudy:1)all
point mutations in the third TPR motif of AIP were the residues tested are essential for the direct interaction of
introduced in different studies (Bell & Poland 2000, Meyer AIP with hsp90; 2) residues p.G272 and p.F288 are also
etal.2000,Laengeretal.2009).Thesestudiesconfirmedthe important for direct AhR binding; 3) the highly conserved
importance of the TPR domain and the specific conserved p.G272residueisrequiredforAIPbindingtoeitherAhRor
aminoacidsintheAIP–AhRandAIP–hsp90interactions. hsp90aloneortotheAhR–hsp90complex;4)allthemutants
Conserved TPR structure
AIP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
TPR2 T P L L L N Y C Q C K L V V E E Y Y E V L D H C S S I L N K Y D D D
Helix A Helix B
AIP
TPR2
Figure 4 Representation of the second TPR motif of AIP. ATPR motif is composed of a pair of antiparallel helices, A and B.
Consensusaminoacidsarelocatedatpositions4,7,8and11inhelixAandatpositions20,24,27and32inhelixB.Residues8and
20arelocatedatthepositionofclosestcontactbetweentheAandtheBhelicesofaTPR,whereasresidue27onhelixBislocatedat
theinterfaceofthreehelices(A,BandtheAhelixofthenextTPRmotif)withinathree-helixbundle(Dasetal.1998).Orangelines
connectinteractingaminoacidsinAandBhelices(modifiedfromJarymowyczetal.(2008)).Patientswithfamilialisolatedpituitary
adenomahavebeenidentifiedwithmutationsaffectingthesecrucialaminoacids,suchasthecytosine(C)atposition8(p.C238Y
(Leontiouetal.2008)),thelysine(K)atposition11(p.K241E;Dalyetal.2007)andtheisoleucine(I)atposition27(p.I257V
(Montananaetal.2009)).
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144 GTRIVELLINANDMKORBONITS AIPinteractingpartners
haveasimilarhalf-lifeinthecelllineused,suggestingthatAIP bothdifferentiationandapoptosis;however,incertaintissues
isstableevenifitisnotassociatedwithanyproteins. such as thyroid and pituitary somatotroph cells, cAMP
Boththep.K266Aandthep.R271Amutantswereshown stimulates cell proliferation (Stork & Schmitt 2002). An
tobeunabletobindhsp90(Bell&Poland2000,Laengeretal. altered cAMP pathway could result in hypertrophy and
2009).Thep.K266Amutantalsoshoweda75–80%reduced hyperplasia of the pituitary, which could lead to the
AhR binding, whereas the 271A–AhR binding was normal developmentofpituitaryadenomas.Forinstance,abnormally
(Bell&Poland2000). high cAMP levels have been linked to CNC and McCune–
As stated previously, in addition to the TPR domain, the Albright syndrome (MAS, MIM# 174800) and have been
five C-terminal amino acids of AIP are also important for identified in 30–40% of sporadic GH-secreting adenomas.
AhR (and probably hsp90) binding. Another proof of their The underlying cause of these high cAMP levels is
crucial role was given by an alanine-scanning mutagenesis constitutively activating mutations in the stimulatory G
experiment,whichdemonstratedthatthereplacementofany protein (Ga, MIM# 139320) in MAS and sporadic
s
ofthelastfouraminoacidswithalanineablatesthebindingto somatotropinomas and inactivating mutations in the protein
theAhR(Bell&Poland2000). kinase A regulatory subunit-1-alpha (PRKAR1A) in CNC
(Boikos&Stratakis2007).
cAMPisgeneratedinthecytoplasmbyactivationofadenylyl
Cytoskeletal proteins
cyclase, but it is inactivated by phosphodiesterases (PDEs).
Afterligand-dependentactivationinthecytoplasm,signalling PDEsareahugefamilyofenzymesthatcatalysethehydrolysis
proteinsthataffectgenetranscription,suchastheAhRorthe ofcAMPandcGMP,generatingthecorrespondingnucleotides
GR,movetotheirsitesofactionwithinthenucleus.Lotsof 50AMPand50GMP.ThePDEsuperfamilycanbesubdivided
evidences point out the essential role of the hsp90-binding into11subfamiliesthatdifferbystructure,enzymaticproper-
immunophilins in mediating various phases of nuclear ties, sensitivity to different inhibitors and specific expression
receptor movements. For instance, FKBP52, which is profiles. Each subfamily comprises from one to four distinct
associated with several steroid receptor heterocomplexes, genes and each gene, in turn, generates several transcripts
has been shown to interact with tubulin cytoskeletal (Bender & Beavo 2006). This multiplicity of PDE isoforms
networks. This interaction takes place through the PPIase (currently more than 50 different PDE proteins with tissue-
domain of FKBP52 and the cytoplasmic dynein, the motor specific subtypes have been identified) ensures the compart-
proteinthatprocessesalongthemicrotubulesinaretrograde mentalisation, fine-tuning and crosstalk of the cAMP and
direction towards the nucleus (Pratt et al. 2004). It is thus cGMP pathways(Zaccolo&Movsesian2007).All the PDEs
conceivable to suppose that AIP could also associate with consist of a modular architecture, with variable regulatory
actinortubulinfilamentsinordertoregulatethecytoplasmic domains located at the N-terminus and a highly conserved
localisationoftheAhR. catalyticregionattheC-terminus(Conti&Beavo2007).
Threestudiesaddressedthismatter.Inone,itwasshownby
aco-immunoadsorptionassay,thatthePPIase-likedomainof PDE4A5ThecAMP-specificPDE4groupformsthelargest
AIPdoesnotbind,orbindsonlyveryweakly,thecytoplasmic PDE subfamily, which is the main enzyme responsible for
dynein (Galigniana et al. 2002). In another study, it was cAMP degradation (Lugnier 2006). PDE4s can be distin-
demonstrated that the well-documented AIP-mediated guishedfromotherPDEsubfamiliesbysequenceidentityin
cytoplasmic retention of the AhR (Kazlauskas et al. 2000, the catalytic region and by the presence of specific regions,
2002, LaPres et al. 2000, Petrulis et al. 2000, Kashuba et al. located at the N-terminal portion of the proteins, called
2006) involves the anchoring of the complex to actin upstream conserved regions 1 and 2 (UCR1 and UCR2;
filaments (Berg & Pongratz 2002). By co-IP experiments, Bolgeretal.2003,deOliveira&Smolenski2009).Atleast35
this interaction was proved to involve a direct binding of splice variants are encoded by four genes (PDE4A–D;
AIPtoactinandalsototakeplaceonlyinthenon-activated Lugnier 2006). Of these, the highly conserved PDE4A4/5
AhRcomplex,becauseTCDDtreatmentinducestherelease isoform (PDE4A5 is the rat homologue of the human
ofthecomplexfromactin(Berg&Pongratz2002).However, PDE4A4)ischaracterisedbyanextendedN-terminalregion
it should be noted that the AIP–actin interaction was involved in subcellular targeting (Bolger et al. 2003).
subsequently rejected by another group (Petrulis et al. PDE4A4/5isexpressedinawidevarietyoftissues,including
2003). The only differencebetween the two studieswas the thepituitary(Mackenzieetal.2008,Lennoxetal.2011),and
celllineusedfortheexperiments.Inabsenceofaconclusive both membrane and cytosolic localisation has been detected
demonstration, thisinteractioncannot beconsidered certain (Huston et al. 2000). PDE4A5 was demonstrated to interact
atthemoment. with the SH3 domains of SRC family tyrosyl kinases
(O’Connell et al. 1996, Beard et al. 2002), with AKAP3
(Bajpaietal.2006)andwithAIP(Bolgeretal.2003).AIPwas
Phosphodiesterases
initiallyidentifiedasadirectbindingpartnerofPDE4A5bya
ThecAMPfunctionsasanintracellularsecondmessenger in Y2H screening of a ratbraincDNAlibrary.Theinteraction
severalsignallingpathways.cAMPoftenactsasapromoterof was subsequently confirmed by a GST pull-down assay and
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AIPinteractingpartners GTRIVELLINANDMKORBONITS 145
was also demonstrated in mammalian cells. In addition, the in vitro b-galactosidase assay was not showing strong
abilityofAIPtobindPDE4A5wasfoundtobeunaffectedby abnormality (Igreja et al. 2010). Clearly, further data are
the concomitant binding of LYN, a member of the tyrosyl needed to understand whether PDE4A5 binding correlates
kinasefamily. withthetumorigenicpropertiesofAIP.
The interaction was proved to be highly specific, because Combining together the results from these and other
otherimmunophilinsorTPR-containingproteinssuchasthe bindingstudies(Bolgeretal.2003,Laengeretal.2009,Igreja
AIP homologue AIPL1, FKBP51 and FKBP52 were unable et al. 2010), the essential role played bythe R271 residue in
to bind PDE4A5 and do not involve other PDE4 isoforms. mediating the binding of AIP with hsp90 and PDE4A5
The domains mediating the interaction weremapped in the emerges. This amino acid is likely to participate in an
TPR region of AIP and in both the unique N-terminal electrostaticinteractionwiththeEEVDandEELDmotifsof
region(aminoacids11–42)andtheUCR2domain(onlythe hsp90 and PDE4A5 respectively (Bolger et al. 2003), and its
PDE4-conserved EELD motif) of PDE4A5. The binding of mutation completely disrupts these interactions. It is also
AIP to PDE4A5 was demonstrated to lead to three distinct interestingtonotethatthep.R271Whumanmutationaffects
functionalconsequences.Firstisareversible,dose-dependent a CpG site and is the second most common mutational
inhibition of PDE4A5 catalytic activity by w60%, which is hotspotdescribedintheAIPgene(Igrejaetal.2010).
directly mediated by the TPR domain of AIP. A second
outcome,alsoleadingtoadecreasedenzymaticactivity,isthe PDE2A3 Three isoforms of PDE2A (PDE2A1, PDE2A2
attenuationofPDE4A5phosphorylationbyPKA.Thethird and PDE2A3), generated from a single gene by alternative
consequence is an increased sensitivity of PDE4A5 to splicing,havebeenclonedfromseveraldifferentspecies.These
rolipram,thePDE4-specificinhibitor. threeisoformsareidenticalexceptfortheN-terminalregions,
TheimpactofseveralmissenseandnonsenseAIPmutations which are responsible for their different subcellular local-
on PDE4A5 binding was studied by a Y2H b-galactosidase isation.Thehumanvariant(PDE2A3)encodesamembrane-
assay (Bolger et al. 2003, Leontiou et al. 2008, Igreja et al. associatedproteinof941aminoacids.PDE2Afunctionsasa
2010). All four naturally occurring truncating mutations homodimerandeachmonomer isformedbyanN-terminal
investigated (p.R81X, p.Q164X, p.Q217X and p.R304X) domain, two tandem GAF domains (GAF-A and -B) and a
completelyabolishtheinteraction(Leontiouetal.2008,Igreja catalyticC-terminaldomain.PDE2Aisabletohydrolyseboth
etal.2010).The12missensemutationsanalysedwereselected cAMPandcGMP,butinthepresenceofcGMP,whichbinds
based on the evolutionarily conserved structurally critical totheallostericGAF-Bdomain,theenzymeisactivatedand
aminoacidslocatedwithintheconsensusTPRdomain(Das increases its affinity for cAMP, resulting in a greater
etal.1998,Russelletal.1999,Scheufleretal.2000)(p.N236A, hydrolysing capacity for cAMP than for cGMP (Bender &
p.K266Aandp.R271A)(Bolgeretal.2003)orbecausethey Beavo 2006). This enzyme thus contributes to the crosstalk
were described in patients or controls (p.R16H, p.V49M, between these two second messenger pathways (Zaccolo &
p.K103R, p.C238Y, p.K241E, p.I257V, p.R271W, p.A299V Movsesian 2007). PDE2A is strongly expressed in the brain
and p.R304Q) (Leontiou et al. 2008, Igreja et al. 2010). with a moderate presence in peripheral tissues such as the
Regarding p.N236A, p.K266A and p.R271A, it was shown adrenal gland and heart and skeletal muscle (Rosman et al.
that only the latter significantly attenuates the interaction of 1997, Sadhu et al. 1999, Stephenson et al. 2009, Lin et al.
AIP with PDE4A5 and also its ability to inhibit PDE4A5, 2010).Inaddition,PDE2Aexpressionhasbeenobservedinrat
whereas the others exhibit normal binding and do not (Velardez et al. 2000, Stephenson et al. 2009) and human
compromise the inhibitory capacity of AIP (Bolger et al. pituitary(Lennoxetal.2011).
2003). The other nine missense variants investigated can be de Oliveira et al.(2007) identified an interaction between
dividedintotwogroups:thefirstgroupcomprisesmutations the human PDE2A and the AIP by a Y2H screening of a
with b-galactosidase assay activity values more than fivefold humanbraincDNAlibrary.Theinteractionwassubsequently
different from WT (p.K103R, p.C238Y, p.K241E and confirmedbyGSTpull-downandco-IPexperimentsbothin
p.R271W), suggesting that they lead to a complete loss of cell lines and in brain tissue lysates. The two proteins were
PDE4A5–AIP binding (Leontiou et al. 2008, Igreja et al. found to colocalise in the cytosol, with the occasional
2010),whereastheothergrouphasnormal(p.V49M)orless involvement of the plasma membrane. The regions that
than threefold different b-galactosidase assay activity values mediatetheinteractionweremappedintheGAF-Bdomain
from WT (p.R16H, p.I257V, p.A299Vand p.R304Q). The ofPDE2AandtheC-terminalhalf(aminoacids170–330)of
low impact of the p.R16H variant on PDE4A5 binding AIP. In addition, preliminary results suggested that AIP was
(Igrejaetal.2010),togetherwithitsnormalinteractionwith abletointeractwithPDE2Aandhsp90atthesametime.
theRETprotein(describedinRETsection;Georgitsietal. DifferentfromwhatwaspreviouslyshownforPDE4A5,the
2007), suggest that if this is a pathogenic variant and not a enzymaticactivityofPDE2AwasunaffectedbyAIPbinding.
rare polymorphism, then based on the in vitro studies, PDE2A,duetoitsbindingtoAIP,islocatedinthevicinityof
a PDE4A5- and RET-independent mechanism may take AhRandisabletolowerthelocalcAMPconcentrationinthe
place. Notably,the p.R304Qvariant has verystrong clinical compartment where the AhR complex is located, therefore
data suggesting that it is a disease-causing variant, but the the TCDD- and especially the forskolin-induced nuclear
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146 GTRIVELLINANDMKORBONITS AIPinteractingpartners
translocation of AhR is 40% (with TCDD) or 55% (with with the GR complex. However, subsequently, co-IPassays
forskolin) lower in PDE2A-transfected cells compared with inamammaliancellline showedthatthisinteractionoccurs
control cells. Furthermore,thisinhibition wasdemonstrated viahsp90(Laengeretal.2009).TheeffectofAIPontheGR
tocorrelatewithareductionoftheAhRfunction,asreported signallingisinhibitoryduetoadelayednuclearaccumulation
byareportergeneassay. of GR after ligand binding, with subsequent decrease of
At present, it is unknown if both PDE4A5 and PDE2A GR’s transcriptional activity (Laenger et al. 2009, Schulke
can simultaneously associate via AIP to the AhR complex et al. 2010). A similar situation has occurred with FKBP51,
(deOliveira&Smolenski2009). which was found not to be associated with GR inyeast but
was associated in mammalian cells. A possible explanation
could be the lack of other TPR domain protein partners of
Nuclear receptors
thereceptorresultinginsuboptimalactivityinyeast(Laenger
Several studies demonstrated the involvement of AIP in etal.2009).
variousnuclear receptorsignallingpathways. Apart from ER and GR, some evidences suggest the
potential of AIP to interact with other steroid hormone
Oestrogen receptor a ERa and ERb mediate the receptors. In particular, AIP has recently been shown to
biological effects of oestrogens both in reproductive and in stronglyinhibitthetranscriptionalactivityofthereceptorsfor
nonreproductive organs in both sexes. After ligand binding, progesterone(PR)andandrogen(AR)(Schulkeetal.2010).
the ERs sequentially dimerise, translocate into the nucleus,
associate with several coregulator proteins, and regulate the Peroxisome proliferator-activated receptor a The
transcriptional activity of oestrogen target genes (Nilsson & peroxisome proliferator-activated receptor a (PPARa,
Gustafsson 2011). ERa and ERb display distinct or even MIM# 170998) is a soluble transcription factor belonging,
oppositeeffects:intumourcells,ERawasshowntopromote as the GR, to the nuclear receptor superfamily. PPARa can
cellproliferation(Zengetal.2008),whereasERbstimulates beactivatedbydifferentlipophiliccompounds,and,inturn,it
apoptosis (Cheng et al. 2004). The physiological actions of associates with the retinoid X receptor a (RXRa). This
oestrogensarethustheresultofabalancebetweenERaand heterodimer activates the transcription of several genes
ERbsignalling(Heldringetal.2007). encoding enzymes involved in the lipid and lipoprotein
It has recently been shown that AIP is involved in ERa metabolism(Yoon2009).
transcriptionalactivationbyinteractingwiththeco-activator Byco-IPexperiments,usinganantibodyhighlyspecificfor
TIF-2 (Pongratz et al. 2009), which is structurally relatedto the PPARa isoform and therefore not binding to the other
members of the bHLH–PAS family (Voegel et al. 1998). In PPARssubtypesPPARbandPPARg,themousePPARawas
particular,AIPnegativelyregulatestheproteinlevelsofTIF-2 found to form a complex with AIP and hsp90 in the liver
both in the presence and in the absence of ligand, thus cytosol. However, as PPARa is predominantly nuclear, the
exertinganegativeeffectonERatranscriptionalactivity.We authorshypothesisedthatthiscomplexcouldexistaswellin
could hypothesise that AIP may have a role in preventing the nucleus (Sumanasekera et al. 2003). Similar to the
ERa-dependenttumourgrowth. inhibition exerted on the transcriptional activity of the
HBVXproteinandGR(Kuzhandaiveluetal.1996,Laenger
Glucocorticoid receptor The GR is a member of the etal.2009),AIPwasfoundtorepressPPARaactivitywhen
nuclear receptor superfamily. Like the AhR, it has been overexpressed (Sumanasekera et al. 2003). However, the
demonstrated to exist in a multiprotein heterocomplex normallylowexpressionlevelsofAIPintheliver,incontrast
containing two molecules of hsp90 and other co-chaperone to the high PPARa expression, suggest that the inhibitory
proteins, such as p23, PP5 and FKBP52 (Grad & Picard effect of AIP might be very weakor not explicated at all in
2007). Moreover, the GR signalling pathway shares some physiologicalconditions.
mechanistic similarities with the AhR pathway. Both the
receptors reside in the cytoplasm in the absence of the Thyroid hormone receptors b1 Thyroid hormone
respective ligands and, on their binding, they undergo receptors (TRs) mediate the genomic actions of the thyroid
conformationalchanges,whichleadtonucleartranslocation. hormone triiodothyronine (T ). TRs are nuclear receptors
3
Inside the nucleus, the GR homodimerises and the dimer derived from two genes, THRA and THRB. The THRB
binds to the recognised enhancer elements, activating the (MIM#190160)geneencodesthreeisoforms,b1,b2andb3
transcription of the target genes (Heitzer et al. 2007). The (Cheng et al. 2010). TRb1 is involved in the negative
similarmechanismofactionwiththeAhRandespeciallythe feedback of T on TRH production in the paraventricular
3
presence of TPR-containing proteins as GR regulators nucleus (PVN) but also mediates the T -independent
3
prompteddifferentgroupstotestwhether AIPispartofthe activation of TRH transcription. As the mechanism by
GRcomplex. which TRb1 exerts this activating role was unknown,
The first studies by Carver et al. (1998), conducted by a Froidevauxetal.(2006)decidedtolookforTRb1-interacting
Y2H screening (Carver & Bradfield 1997) and by co-IP proteins.UsingaY2HassaytoscreenamousePVNcDNA,
experimentsinyeast,demonstratedthatAIPdoesnotinteract AIP was identified as a new TRb1 partner. AIP and TRb1
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