Practice Guidelines for the Perioperative Management of

137

REVIEW

AIP and its interacting partners

Giampaolo Trivellin1,2 and Marta Korbonits1

1Department of Endocrinology, Bart’s and the London School of Medicine, Queen Mary University of London, London EC1M 6BQ, UK2Endocrinology Division, Department of Medical and Surgical Sciences, Via Ospedale, 105, 35128 Padova, Italy

(Correspondence should be addressed to M Korbonits; Email: [email protected])

Abstract

Germline mutations in the aryl hydrocarbon receptor-

interacting protein gene (AIP) predispose to young-onset

pituitary tumours, most often to GH- or prolactin-secreting

adenomas, and most of these patients belong to familial

isolated pituitary adenoma families. The molecular pathway

initiated by the loss-of-function AIP mutations leading to

pituitary tumour formation is unknown. AIP, a co-chaper-

one of heat-shock protein 90 and various nuclear receptors,

belongs to the family of tetratricopeptide repeat (TPR)-

containing proteins. It has three antiparallel a-helix motifs

Journal of Endocrinology (2011) 210, 137–1550022–0795/11/0210–137 q 2011 Society for Endocrinology Printed in Great

(TPR domains) that mediate the interaction of AIP with

most of its partners. In this review, we summarise the

known interactions of AIP described so far. The

identification of AIP partners and the understanding of

how AIP interacts with these proteins might help to explain

the specific phenotype of the families with heterozygous

AIP mutations, to gain deeper insight into the pathological

process of pituitary tumour formation and to identify novel

drug targets.

Journal of Endocrinology (2011) 210, 137–155

Introduction

Familial pituitary adenomas can occur in three diseases:

multiple endocrine neoplasia type 1 (MEN1, MIM#

131100), the rare Carney complex (CNC, MIM# 160980)

and the recently described familial isolated pituitary adenoma

(FIPA, MIM# 102200). In the first two syndromes multiple

endocrine organs are involved, whereas in FIPA tumours

occur only in the pituitary gland (Chahal et al. 2010). A locus

has previously been identified in a phenotypically relatively

well-defined subgroup of FIPA families, but the gene causing

the disease, namely the aryl hydrocarbon receptor-interacting

protein gene (AIP, MIM# 605555), had not been identified

until 2006 (Benlian et al. 1995, Gadelha et al. 2000, Soares

et al. 2005, Vierimaa et al. 2006). About 30% of all FIPA

families and 50% of acromegaly families have a mutation in

the AIP gene (Chahal et al. 2010). Families with AIP

mutations have a characteristic phenotype: childhood- or

young-onset disease (the mean age at diagnosis is around 20

years), primarily GH- or prolactin-secreting adenomas (the

majority of the patients have GH- or mixed GH- and

prolactin-secreting adenomas, a minority have prolactinomas,

whereas other pituitary adenoma types are rarely observed),

and large and invasive pituitary tumours that do not respond

well to somatostatin analogue treatment (Leontiou et al. 2008,

Daly et al. 2010). Pituitary apoplexy can be a presenting

feature in patients with AIP mutation (Chahal et al. 2011).

Occasionally, patients with young-onset acromegaly or other

childhood-onset pituitary adenomas carry germline mutation

in the AIP gene without apparent family history (Cazabat

et al. 2007, Georgitsi et al. 2008, Daly et al. 2010, Stratakis

et al. 2010). The penetrance of the disease is low, probably

around 30%, and there is a male preponderance (Cain et al.

2010, Daly et al. 2010). Three-quarters of AIP mutations lead

to a truncated protein, and some of the missense mutations

have also shown to result in loss of function of the protein

suggesting, together with loss of heterozygosity data, that

AIP functions as a tumour suppressor gene in the pituitary

gland (Soares et al. 2005, Vierimaa et al. 2006, Leontiou et al.

2008, Igreja et al. 2010).

At first, AIP seems to be an unusual gene causing

pituitary adenoma as it was previously only known as a

co-chaperone of nuclear receptors or viral proteins

(Kuzhandaivelu et al. 1996, Carver & Bradfield 1997, Ma

& Whitlock 1997, Meyer et al. 1998, Kashuba et al. 2000).

AIP, also known as X-associated protein-2 (XAP2;

Kuzhandaivelu et al. 1996), Ah receptor-activated 9

(ARA9; Carver & Bradfield 1997) or FK506-binding

protein 37 (FKBP37; Blatch et al. 2006), is a 37 kDa

cytoplasmic protein. Structurally, it shares a significant

degree of homology with immunophilins, such as FKBP52

(52 kDa FK506-binding protein), as it has a peptidyl-prolyl

DOI: 10.1530/JOE-11-0054Britain Online version via http://www.endocrinology-journals.org

Downloaded from Bioscientifica.com at 12/11/2019 05:22:57AMvia free access

http://dx.doi.org/10.1530/JOE-11-0054

G TRIVELLIN AND M KORBONITS . AIP interacting partners138

cis–trans isomerase (PPIase)-like domain. Immunophilins

are a huge family of ubiquitous and conserved proteins,

which possess PPIase domains that bind immunosuppressant

drugs of the FK506 or of the cyclosporin A groups (Galat

& Metcalfe 1995). However, AIP does not function as an

immunophilin. AIP lacks affinity for the immunosuppres-

sant drugs FK506 and rapamycin and the PPIase-like

domain displays no enzymatic activity (Carver et al. 1998,

Laenger et al. 2009), so AIP cannot be considered a true

immunophilin. These data are consistent with a weak

homology between the PPIase domains of AIP and FKBP12

(only five of the 14 amino acids of the FK506-binding domain

of FKBP12 are conserved in AIP (Carver et al. 1998)) and

explain the different biochemical properties of AIP.

AIP belongs to the family of tetratricopeptide repeat

(TPR) domain-containing proteins, such as the aryl

hydrocarbon receptor-interacting protein-like 1 (AIPL1),

protein phosphatase 5 (PP5), FK506-binding protein 51

(FKBP51), FKBP52, cyclophilin 40 (Cyp40), carboxyl

terminus of Hsc70-interacting protein (CHIP), and heat-

shock protein 70 (Hsp70)/Hsp90 organising protein (Hop;

D’Andrea & Regan 2003), and has three TPR motifs and a

final a-7 helix at the C-terminus (Fig. 1). TPR domains are

highly degenerate consensus sequences of 34 amino acids,

often arranged in tandem repeats, formed by two a-helices

forming an antiparallel amphipathic (having both hydrophilic

and lipophilic properties) structure that mediates intra- and

inter-molecular interactions in many proteins (Goebl &

Yanagida 1991).

The AIP protein sequence is evolutionarily conserved

among species. The protein sequence of human AIP is 100,

94 and 93% identical to chimpanzee, mouse and rat AIP

respectively (Supplementary Table 1, see section on supple-

mentary data given at the end of this article). Furthermore,

AIP is located on a conserved synthetic block in the human,

mouse and rat genome (Fig. 2). The fact that AIP is a highly

conserved protein could be expected for two reasons: first,

because AIP is associated with a human disease, and several

studies have found that genes causing human disease are more

conserved than non-disease genes (Lovell et al. 2009), and

secondly, because AIP has been demonstrated to be essential

in cardiac development and in maintaining productive

erythropoiesis in mice (Lin et al. 2007, Kang et al. 2011)

and previous studies in mammals and other eukaryotes

showed that essential genes are usually located in highly

conserved genomic regions (Lovell et al. 2009).

As expected from the presence of TPR motifs in the AIP

protein, several proteins have been identified which interact

with AIP. The identification of these molecules and the

understanding of how AIP interacts with them could give

us an insight into the pathological process of pituitary

tumour formation and may lead to new therapeutic targets.

The main focus of this review is thus to summarise the

AIP-interacting partners described so far. The functional

studies available about specific AIP amino acid mutations

are also reviewed.


AIP-interacting proteins

Viral proteins

Hepatitis B virus X protein AIP was originally described

as a protein associated with the X protein of the hepatitis B

virus (HBV; Kuzhandaivelu et al. 1996), a small human DNA

virus that causes acute and chronic hepatitis. Among the few

genes contained in the genome of HBV, there is an open

reading frame that encodes a 154 amino acid regulatory

protein, termed X protein. This protein, which does not have

a human homologue, activates the transcription of a wide

variety of different genes through interaction with cellular

factors (Koike 2009). In order to identify new proteins that

may interact with X, the authors used the yeast two-hybrid

(Y2H) method. Among the several potential cDNAs coding

the protein X binding protein, six were found to be

overlapping clones of a full-length cDNA encoding the

same gene. The gene was named XAP2 because it was the

second protein found to interact with the HBV X protein by

this technique. The full-length AIP cDNA was subsequently

isolated and in vitro translated in a rabbit reticulocyte lysate.

The translated product corresponded perfectly with the native

AIP from HeLa cells and had an apparent molecular mass of

36 kDa. AIP RNA expression was evaluated and detected in

several different tissues and cell lines, but very low levels were

found in the liver. The ubiquitous expression of AIP was

subsequently confirmed in human and murine tissues at the

mRNA and protein level (Kuzhandaivelu et al. 1996, Ma &

Whitlock 1997, Carver et al. 1998, Meyer et al. 1998, 2000,

Yano et al. 2003).

The X protein–AIP interaction was also demonstrated

to occur in vitro by testing the ability of a glutathione

S-transferase (GST)–X fusion protein to bind to a 35S-labelled

AIP. Using different X mutants, the interaction with AIP was

then shown to be mediated by X protein residues 13–26, a

region highly conserved among all mammalian hepadnaviruses.

Further evidence regarding this interaction shows similar

cytoplasmic distributions of X protein and AIP with immuno-

cytochemistry in X protein-transfected mammalian cells.

Overexpressing AIP resulted in inhibited X protein

transcriptional activity, suggesting that AIP is an important

negative regulator of the X protein and that their interaction

may play a role in HBV pathology.

EBNA-3 EBV-immortalised lymphoblastoid cell lines

express, among others, six nuclear antigens (EBNA 1–6)

and three latent membrane proteins (LMP 1, 2a, 2b), whose

concerted action is essential for the immortalisation and

transformation of B-cells (Tomkinson et al. 1993). As the

identification of cellular proteins that can interact with the

transformation associated EBNAs was not yet complete,

Kashuba et al. (2000) searched for proteins that can bind to the

transcriptional regulator EBNA-3, which again does not have

a human homologue, in a Y2H system. Among the several

clones identified, one corresponded to the AIP protein.

www.endocrinology-journals.org


http://joe.endocrinology-journals.org/cgi/content/full/JOE-11-0054/DC1

A

B

31 121 179 212 231 264 298 330

PPIase-like TPR1 TPR2 TPR3 α-7

R16H V49M K103R N236A K241E

C238Y I257V

K266A

Y268A

G272D/G272E

R271W

A284T

F288A

E293G

A299V

R304Q

TPR1 TPR2

TPR3 α-7

PPIase-like

Figure 1 (A) Schematic structure of the AIP protein. Structurally, AIP is similar to immunophilins. AIP contains a PPIase-like domain at theN-terminus, which shows a weak identity to the low molecular weight FKBP12 (12 kDa FK506-binding protein) (Carver & Bradfield 1997),but does not show immunophilin activity (Carver et al. 1998, Laenger et al. 2009). The C-terminal part of the molecule contains three TPRdomains that are conserved 34 amino structures consisting of two antiparallel a helices. There is a terminal a-7 helix that is crucial forprotein–protein interaction. Numbers underneath the protein structure represent amino acids. Boxes with different shapes representdifferent domains. The location of the missense mutations investigated in functional binding assays is marked. PPIase-like, peptidyl-prolylcis–trans isomerase-like domain; TPR, tetratricopeptide repeat domain. (B) Hypothetical structure of AIP based on the crystal structure ofthe related protein FKBP51 (Igreja et al. 2010). The PPIase-like domain, the three TPR motifs with three pairs of anti-parallel a-helices andthe final extended a-helix, a-7, are highlighted. The amino acids subjected to site-directed mutagenesis analyses to study their role in theAhR complex are shown in red (Bell & Poland 2000, Meyer et al. 2000, Laenger et al. 2009).

AIP interacting partners . G TRIVELLIN AND M KORBONITS 139

Subsequently, this interaction was also confirmed in vitro using

a GST pull-down assay.

Concordant to that shown for the aryl hydrocarbon

receptor (AhR (MIM# 600253); see AIP–AhR–Hsp90

section), AIP was also found to translocate to the nucleus

on expression of EBNA-3. The authors hypothesised that

as AIP can bind to the transforming proteins of two

evolutionarily distant viruses and also to the AhR, a

phylogenetically ancient protein conserved in vertebrates

and invertebrates (Hahn 2002), the AhR signal transduction

pathway could be involved in virus-induced cell transfor-

mation. This hypothesis was further supported by a following

demonstration from the same group that besides AIP, EBNA-

3 can also directly interact with the AhR, with AIP enhancing

the stability of the complex. As a result of this association,

an enhanced transcription of AhR-responsive genes has been

observed (Kashuba et al. 2006).


AIP–AhR–Hsp90 complex

More or less parallel to the discovery of the binding with

HBV X protein (Kuzhandaivelu et al. 1996), AIP was

independently identified by three laboratories to also interact

with two other proteins: the AhR (Carver & Bradfield 1997,

Ma & Whitlock 1997, Meyer et al. 1998) and the hsp90

(Carver et al. 1998, Meyer & Perdew 1999, Bell & Poland

2000, Kazlauskas et al. 2002, Yano et al. 2003, Laenger et al.

2009, Schulke et al. 2010).

AIP–AhR The AhR, a basic helix–loop–helix protein

of the Per–ARNT–Sim (PAS) family of transcriptional

regulators, is a cytoplasmic transcription factor that can be

activated by a wide variety of structurally diverse exogenous

ligands, the prototype of which is the 2,3,7,8-tetrachloro-

dibenzo-p-dioxin (TCDD), as well as by some endogenous



206730K

206725K

206720K

206715K

206710K

206705K

206700K

1

206695K

206690K

206685K

LOC688884

Gpr152

11:67,195,935-67,202,879 (+) RPS6KB2

PTPRCAP

CORO1B

GPR152

CABP4

TMEM134

AIP

PITPNM1

CDK2AP2

CABP2

NDUFV1

Rps6kb2 19:4,156,977-4,163,245 (–)

19:4,154,646-4,156,710 (+)

19:4,148,663-4,154,035 (+)

19:4,139,799-4,145,741 (+)

19:4,135,423-4,139,609 (–)

19:4,125,960-4,132,307 (+)

19:4,114,446-4,125,827 (–)

19:4,100,622-4,113,961 (+)

19:4,097,351-4,099,017 (+)

19:4,083,490-4,087,340 (+)

19:4,007,505-4,012,725 (–)

19

19

19

19

19

19

19

19

19

19

19

Ptprcap

Coro1b

Gpr152

Cabp4

Tmem134

Aip

Pitpnm1

Cdk2ap2

Cabp2

Ndufv1

11:67,202,981-67,205,153 (–)

11:67,205,518-67,211,263 (–)

11:67,218,772-67,220,200 (–)

11:67,222,818-67,226,691 (+)

11:67,231,819-67,236,731 (–)

11:67,250,505-67,258,579 (+)

11:67,259,239-67,272,843 (–)

11:67,273,968-67,276,102 (–)

11:67,286,418-67,290,899 (–)

11:67,374,323-67,380,012 (+)

GPR152

CABP4

TMEM134

AIP

PITPNM1

CDK2AP2

4145K

4140K

4135K

4130K

4125K

4120K

4115K

4110K

4105K

4100K

4095K

19

Cabp4

Tmem134

Aip

Pitpnm1

Cdk2ap2

Cabp4_pr..

RGD13058..

Aip

Pitpnm1

LOC688405

Ratchr 1q2

Mousechr 19qA

Humanchr 11q13

Humanposition

HumanGene

Mousechromosome

Mousegene

Mouseposition

Figure 2 Human–mouse–rat synteny map around the AIP locus (highlighted in red) (http://www.ncbi.nlm.nih.gov/projects/homology/maps/human/chr11/).


compounds, such as the cAMP (Denison & Nagy 2003,

Oesch-Bartlomowicz et al. 2005, Nguyen & Bradfield 2008).

Ligand-free AhR in the cytoplasm binds to two molecules of

hsp90 (Perdew 1988) and the co-chaperone proteins p23

(Nair et al. 1996) and AIP (Carver & Bradfield 1997, Ma &

Whitlock 1997, Meyer et al. 1998). The interaction with

hsp90 shapes the AhR’s ligand-binding domain into a state

competent for ligand binding, and it also negatively regulates

AhR until ligand binding occurs (Beischlag et al. 2008). p23

is part of the AhR complex through interaction with hsp90

(Nair et al. 1996) and its presence is thought to stabilise the

complex (Kazlauskas et al. 1999) and to favour its nuclear

import (Kazlauskas et al. 2001). These functions are in line

with the modulating role that p23 has been shown to exert

in different steroid hormone receptors (Smith et al. 1995,

Dittmar et al. 1997, Freeman et al. 2000). The presence of p23

in the AhR complex seems, however, not to be essential for

the AhR physiology (Cox & Miller 2004, Flaveny et al. 2009).

Using the Y2H method with AhR as the bait, a human

cDNA (termed at the time ARA9; Carver & Bradfield 1997)

and a murine cDNA (termed AIP) were described (Ma &

Whitlock 1997), both shown to be identical to XAP2. At the

same time, since an uncharacterised protein of about 43 kDa

was found to be part of the AhR–hsp90 complex (Chen &

Perdew 1994), a third group decided to identify this protein.

After purification from simian COS-1 cells, the molecular

mass was reassigned from 43 to 38 kDa and the protein

sequence was found to be 98% identical to human AIP

(Meyer et al. 1998).

There is considerable controversy regarding the effect of

AIP on AhR function. Some studies reported that AIP can

enhance the transcriptional activity and expression levels of


the AhR (Carver & Bradfield 1997, Ma & Whitlock 1997,

Meyer et al. 1998, 2000, LaPres et al. 2000, Nukaya et al.

2010), whereas others described an inhibitory function

(Hollingshead et al. 2004, Pollenz & Dougherty 2005, Pollenz

et al. 2006). This variability is due to several factors, including

species- and tissue-specific differences. Apart from these

conflicting results, AIP was repeatedly shown to protect the

AhR from ubiquitin-dependent degradation through the

proteasome (Kazlauskas et al. 2000, Morales & Perdew 2007).

Consistent with that, low levels or loss of AIP correlates with

low expression of AhR (Jaffrain-Rea et al. 2009).

After ligand binding, AhR undergoes a conformational

change that exposes a nuclear localisation sequence, resulting

in translocation of the complex into the nucleus, where it

forms a heterodimer with the aryl hydrocarbon receptor

nuclear translocator 1 (ARNT, also known as HIF1b, MIM#

126110), which leads to the activation of AhR-sensitive genes.

In mice, ligand binding leads to the dissociation of AIP from

the complex as it enters the nucleus (Ma & Whitlock 1997),

whereas in humans the association is maintained in the

nucleus (Carver & Bradfield 1997, Ramadoss et al. 2004).

In addition to its well-established role as a transcription

factor, the ligand-activated AhR has also been shown to

modulate the functions of other transcription factors, such as

the oestrogen receptor (ERa and ERb) and the androgen

receptor (AR). The crosstalk of AhR with ER and AR

explicates via a direct association in the nucleus, which

modulates oestrogen/androgen signalling both positively

and negatively depending on the cellular context (Ohtake

et al. 2009, Pongratz et al. 2009). Furthermore, a novel and

unexpected role for AhR as a ligand-dependent E3 ubiquitin

ligase has also recently been described: the ligand-bound



http:\\http://www.ncbi.nlm.nih.gov/projects/homology/maps/human/chr11/

http:\\http://www.ncbi.nlm.nih.gov/projects/homology/maps/human/chr11/


AhR promotes the ubiquitination and proteasomal

degradation of ER and AR through the assembly of a

ubiquitin ligase complex, referred to as CUL4BAhR. AIP,

however, seemed unlikely to influence the CUL4BAhR-

mediated ER degradation (Ohtake et al. 2007).

AIP–Hsp90 Hsp90 is a highly abundant molecular

chaperone, which associates as a dimer with a set of highly

different client proteins. Hsp90 is required to maintain

signalling proteins in an active conformation that can be

rapidly triggered by ligands. Hsp90 functions as the core

component of a dynamic set of multiprotein complexes,

involving a set of co-chaperones. Structurally, hsp90 can be

divided into five domains: a highly conserved N-terminal

domain involved in nucleotide and drug binding, a charged

domain, a middle domain with ATPase activity involved in

client protein binding, a second charged domain and a

C-terminal domain involved in dimerisation and binding of

TPR-containing proteins (mediated by a conserved EEVD

motif) (Pearl & Prodromou 2006).

The direct but moderate association of AIP with hsp90 has

been demonstrated in different studies (Carver et al. 1998,

Meyer & Perdew 1999, Bell & Poland 2000, Kazlauskas et al.

2002, Yano et al. 2003, Laenger et al. 2009, Schulke et al.

2010). Hyperacetylation of hsp90 was found to lead to the loss

of complex formation with AhR, p23 and AIP (Kekatpure

et al. 2009). Discordant findings arose about the role exerted

by hsp90 in assisting the AIP–AhR interaction. A study

demonstrated that AhR, in order to bind AIP, needs to fold

into the mature ligand-binding conformation with the help

of hsp90 (Bell & Poland 2000). This requirement of hsp90

was instead proved to be not essential in another report

(Meyer & Perdew 1999).

AIP–Hsc70 In the absence of AhR, AIP was shown to

interact – with higher affinity – with another heat-shock

protein, the heat-shock cognate 70 (hsc70, MIM# 600816),

rather than to hsp90 (Yano et al. 2003). Hsc70 is a

constitutively expressed co-chaperone protein that is

involved, as hsp90, in protein folding and in mitochondrial

protein import (discussed later in Translocase of the outer

membrane of mitochondria 20 (TOMM20) and mito-

chondrial preproteins section) (Young et al. 2003), but it

also functions as an ATPase in the disassembly of clathrin-

coated vesicles during transport of membrane components

through the cell (Alberts et al. 2002). Hsc70 is a member

of the hsp70 family. Although human hsc70 shares 85%

sequence identity with human hsp70, they play different

cellular functions (Gething & Sambrook 1992, Goldfarb et al.

2006). Consistent with this, AIP was found to be unable

to bind hsp70 (Schulke et al. 2010).

AIP interactions with other proteins of the AhRsignalling pathway Apart from AhR and hsp90, it was

also investigated whether AIP binds to two other proteins

involved in the AhR pathway, ARNT and p23.


ARNT was thought to be a good candidate because it

belongs to the same family of transcription factors of AhR and

both share a similar modular structure. However, the results

from three different studies demonstrated that AIP is excluded

from the AhR–ARNT heterocomplex in vitro and in vivo

(Carver & Bradfield 1997, Ma & Whitlock 1997, Meyer &

Perdew 1999). These findings agree with the AhR mapping

data because AIP and ARNT have been shown to contact,

at least in part, the same or an adjacent binding site on the

AhR (Meyer & Perdew 1999). Even if AIP does not interact

with ARNT, two recent studies showed that the expression

of ARNT protein is significantly reduced in AIP-mutated

pituitary tumours, suggesting that loss of AIP leads to

an imbalance in the AhR–ARNT complex formation

(Heliovaara et al. 2009, Raitila et al. 2010).

p23 was then demonstrated to contact AIP in co-immu-

noprecipitation (co-IP) experiments (Hollingshead et al.

2004), but only indirectly via hsp90, as previously demon-

strated for AhR (Nair et al. 1996). AIP was also shown to be

able to displace p23 from the AhR complex, an effect only

specific for the AhR complex and not for hsp90 alone

(Hollingshead et al. 2004).

AIP self-association An interesting finding was the

evidence that AIP can exist in multimeric complexes of at

least two molecules even without requiring AhR or hsp90

(Hollingshead et al. 2004). The self-association of AIP is very

likely mediated by the TPR domain, as demonstrated for

other TPR-containing proteins (Das et al. 1998, Taylor et al.

2001, Nyarko et al. 2007). This suggests that AIP can

homodimerise without the association of other auxiliary

proteins or at least others than AhR or hsp90 and also that

more than one molecule of AIP could be present in the AhR

complex. However, two previous studies, using different

stoichiometric approaches to examine the AhR complex

subunits composition, showed a AhR:hsp90:AIP ratio of

1:2:1 (Chen & Perdew 1994, Petrulis et al. 2000). If these

ratios are correct, the authors suggested that the multimeric

complexes of AIP act as a reservoir able to regulate the

amount of available monomeric AIP that can be included into

the AhR complex (Hollingshead et al. 2004) or maybe in the

other complexes with which AIP has been shown to interact.

Furthermore, the TPR-mediated self-association of AIP

might be a mechanism to specifically regulate its biological

functions, as reported for the TPR-containing proteins

PP5 and Sgt1 (Yang et al. 2005, Nyarko et al. 2007). For

example, the phosphatase activity of PP5 is suppressed by an

autoinhibited conformation maintained by the TPR domain–

catalytic domain interaction (Yang et al. 2005).

Domains mediating AIP–AhR–Hsp90 interactionand functional studies Different studies contributed to

define the domains involved in AIP–AhR–Hsp90 interaction

and also, more specifically, the AIP residues essential for AhR

and hsp90 binding.




Mapping experiments using AIP deletion mutants showed

that the C-terminal half of AIP (residues 154–330), which

contains the three TPR motifs, was necessary for binding

both the AhR and the hsp90 (Fig. 3; Carver et al. 1998, Meyer

& Perdew 1999, Kazlauskas et al. 2000). An indispensable role

in mediating AhR binding was shown for the a-helical

C-terminus (a-7) of murine AIP: deletion of the last five

amino acids abolishes almost completely AhR binding,

without affecting AIP–hsp90 interaction (Bell & Poland

2000). However, another study presented evidences

suggesting that the human final a-7 helix is more likely to

bind hsp90 than AhR (Kazlauskas et al. 2002). These

contrasting results might be explained by species-specific

differences. There are controversial results regarding the role

played by the N-terminus of AIP. Some reports demonstrated

that this region did not interact with either AhR or hsp90

(Carver et al. 1998, Meyer & Perdew 1999, Kazlauskas et al.

2000), whereas Kazlauskas et al. (2002) showed that the

N-terminus contains an additional site of interaction with the

AhR complex. Moreover, the N-terminal part of AIP was

shown to confer stability to the complex and to be essential in

the regulation of the subcellular localisation of AhR

(Kazlauskas et al. 2002). Taken together, these results suggest

that despite the C-terminal region of AIP being capable of

interacting alone with the AhR complex, this interaction is

not functional. This hypothesis is further supported by the

finding that an AIP mutant lacking the first 17 amino acids,

AhR

hsp90

AIP PPIase-like TP

hsp90 N-terminus Chargeddomain

N-terminus Chargeddomain

bHLH PAS A PAS B

Figure 3 Location of key interacting domains of the AIP–AhR–Hsp90 cto amino acid number. bHLH, basic helix–loop–helix; PAS, Per–ARN


even if it did not loose the ability to bind hsp90, was expressed

at lower levels compared with the wild type (WT) protein,

maybe as a result of a higher turnover in cells (Meyer &

Perdew 1999).

In the reciprocal mapping analyses, the boundaries of

the AhR protein segment interacting with AIP were defined

to be approximately between amino acids 380 and 419,

which encompass the C-terminal portion of the PAS

domain (PAS-B; Carver et al. 1998, Meyer & Perdew 1999,

Kazlauskas et al. 2000). This interaction seems to be mediated

by nonpolar or hydrophobic amino acids (Hollingshead et al.

2004). It was also established that AIP, like other immuno-

philins found in hsp90 complexes, binds to the C-terminal

segment of hsp90 (residues 629–732), whereas the AhR binds

to the middle region (residues 272–617) (Meyer & Perdew

1999, Bell & Poland 2000). Hsp90 was found to interact

with two spatially distinct motifs of the AhR, the PAS-B and

the bHLH domains (Antonsson et al. 1995). The domains

involved in the AIP–hsc70 interaction were not experi-

mentally determined (Yano et al. 2003).

Mutational analyses of some TPR domains indicate that

they perform essential functions (Chen et al. 1996, Blom et al.

2004) and the interaction with hsp90 through TPR domains

has been shown to be conserved in plants and animals

(Owens-Grillo et al. 1996). It is, therefore, very likely that this

is a basic protein interaction critical to the function of AIP.

Some studies addressed this issue analysing how mutations in

R1 TPR2 TPR3 α-7

C-terminusMiddle domain Chargeddomain

C-terminusMiddle domain Chargeddomain

Q-rich

omplex. The size of domains is drawn to linear scale proportionalT–Sim homology domain; Q, glutamin-rich domain.




the TPR domain of AIP affect AhR and hsp90 binding, as

well as PDE4A5 and RET (rearranged during transfection;

described in PDE4A5 and RET section).

Site-directed mutagenesis studies in the binding groove of

the TPR domain of PP5, an immunophilin co-chaperone

protein present in the glucocorticoid receptor (GR, MIM#

138040) complex (Silverstein et al. 1997), confirmed the

prediction made from the previously discovered three-

dimensional structure of the protein (Das et al. 1998) that

basic amino acid residues in this region are important for hsp90

binding (Russell et al. 1999). As these residues are conserved in

the TPR domains of other hsp90-interacting proteins, such as

FKBP51, FKBP52 and AIP, they are likely to be of functional

significance in order to mediate efficient interactions with their

binding partners (Fig. 4). Based mainly on this assumption,

point mutations in the third TPR motif of AIP were

introduced in different studies (Bell & Poland 2000, Meyer

et al. 2000, Laenger et al. 2009). These studies confirmed the

importance of the TPR domain and the specific conserved

amino acids in the AIP–AhR and AIP–hsp90 interactions.

101 2 3 4 5 6 7 8 9 11 12 13 14 15 16 1

T P L L L N Y C Q C K L VV E E YAIP

TPR2

AIPTPR2

Helix A

Conserved TPR s

Figure 4 Representation of the second TPR motif of AIP. A TPRConsensus amino acids are located at positions 4, 7, 8 and 11 in heli20 are located at the position of closest contact between the A and ththe interface of three helices (A, B and the A helix of the next TPR mconnect interacting amino acids in A and B helices (modified from Jaadenoma have been identified with mutations affecting these cruci(Leontiou et al. 2008)), the lysine (K) at position 11 (p.K241E; Daly(Montanana et al. 2009)).


The functional consequences of p.Y268A, p.G272D,

p.272E, p.A284T and p.F288A AIP mutants on hsp90 and

AhR binding were studied by Meyer et al. (2000) in the

presence of both proteins or in the absence respectively, of

one of them. In COS-1 cells (AhR-defective cells), each

mutant was impaired in its ability to associate with hsp90,

with only p.Y268A showing a residual level of binding,

compared with the others in which no binding at all was

observed. Vice versa, in the absence of hsp90, it was found

that only the p.Y268A and the p.A284T mutations were able

to bind to the AhR, whereas the others were not. In the

presence of the AhR–hsp90 complex, it was then found that

all the AIP mutants can bind AhR–hsp90 complex except the

p.G272D and the p.G272E mutants. The following four

conclusions can be drawn from the results of this study: 1) all

the residues tested are essential for the direct interaction of

AIP with hsp90; 2) residues p.G272 and p.F288 are also

important for direct AhR binding; 3) the highly conserved

p.G272 residue is required for AIP binding to either AhR or

hsp90 alone or to the AhR–hsp90 complex; 4) all the mutants

7 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

EY V L D H C S S I L N K Y D D D

Helix B

tructure

motif is composed of a pair of antiparallel helices, A and B.x A and at positions 20, 24, 27 and 32 in helix B. Residues 8 ande B helices of a TPR, whereas residue 27 on helix B is located atotif) within a three-helix bundle (Das et al. 1998). Orange linesrymowycz et al. (2008)). Patients with familial isolated pituitaryal amino acids, such as the cytosine (C) at position 8 (p.C238Yet al. 2007) and the isoleucine (I) at position 27 (p.I257V




have a similar half-life in the cell line used, suggesting that AIP

is stable even if it is not associated with any proteins.

Both the p.K266A and the p.R271A mutants were shown

to be unable to bind hsp90 (Bell & Poland 2000, Laenger et al.

2009). The p.K266A mutant also showed a 75–80% reduced

AhR binding, whereas the 271A–AhR binding was normal

(Bell & Poland 2000).

As stated previously, in addition to the TPR domain, the

five C-terminal amino acids of AIP are also important for

AhR (and probably hsp90) binding. Another proof of their

crucial role was given by an alanine-scanning mutagenesis

experiment, which demonstrated that the replacement of any

of the last four amino acids with alanine ablates the binding to

the AhR (Bell & Poland 2000).

Cytoskeletal proteins

After ligand-dependent activation in the cytoplasm, signalling

proteins that affect gene transcription, such as the AhR or the

GR, move to their sites of action within the nucleus. Lots of

evidences point out the essential role of the hsp90-binding

immunophilins in mediating various phases of nuclear

receptor movements. For instance, FKBP52, which is

associated with several steroid receptor heterocomplexes,

has been shown to interact with tubulin cytoskeletal

networks. This interaction takes place through the PPIase

domain of FKBP52 and the cytoplasmic dynein, the motor

protein that processes along the microtubules in a retrograde

direction towards the nucleus (Pratt et al. 2004). It is thus

conceivable to suppose that AIP could also associate with

actin or tubulin filaments in order to regulate the cytoplasmic

localisation of the AhR.

Three studies addressed this matter. In one, it was shown by

a co-immunoadsorption assay, that the PPIase-like domain of

AIP does not bind, or binds only very weakly, the cytoplasmic

dynein (Galigniana et al. 2002). In another study, it was

demonstrated that the well-documented AIP-mediated

cytoplasmic retention of the AhR (Kazlauskas et al. 2000,

2002, LaPres et al. 2000, Petrulis et al. 2000, Kashuba et al.

2006) involves the anchoring of the complex to actin

filaments (Berg & Pongratz 2002). By co-IP experiments,

this interaction was proved to involve a direct binding of

AIP to actin and also to take place only in the non-activated

AhR complex, because TCDD treatment induces the release

of the complex from actin (Berg & Pongratz 2002). However,

it should be noted that the AIP–actin interaction was

subsequently rejected by another group (Petrulis et al.

2003). The only difference between the two studies was the

cell line used for the experiments. In absence of a conclusive

demonstration, this interaction cannot be considered certain

at the moment.

Phosphodiesterases

The cAMP functions as an intracellular second messenger in

several signalling pathways. cAMP often acts as a promoter of


both differentiation and apoptosis; however, in certain tissues

such as thyroid and pituitary somatotroph cells, cAMP

stimulates cell proliferation (Stork & Schmitt 2002). An

altered cAMP pathway could result in hypertrophy and

hyperplasia of the pituitary, which could lead to the

development of pituitary adenomas. For instance, abnormally

high cAMP levels have been linked to CNC and McCune–

Albright syndrome (MAS, MIM# 174800) and have been

identified in 30–40% of sporadic GH-secreting adenomas.

The underlying cause of these high cAMP levels is

constitutively activating mutations in the stimulatory G

protein (Gas, MIM# 139320) in MAS and sporadic

somatotropinomas and inactivating mutations in the protein

kinase A regulatory subunit-1-alpha (PRKAR1A) in CNC

(Boikos & Stratakis 2007).

cAMP is generated in the cytoplasm by activation of adenylyl

cyclase, but it is inactivated by phosphodiesterases (PDEs).

PDEs are a huge family of enzymes that catalyse the hydrolysis

of cAMP and cGMP, generating the corresponding nucleotides

50AMP and 50GMP. The PDE superfamily can be subdivided

into 11 subfamilies that differ by structure, enzymatic proper-

ties, sensitivity to different inhibitors and specific expression

profiles. Each subfamily comprises from one to four distinct

genes and each gene, in turn, generates several transcripts

(Bender & Beavo 2006). This multiplicity of PDE isoforms

(currently more than 50 different PDE proteins with tissue-

specific subtypes have been identified) ensures the compart-

mentalisation, fine-tuning and crosstalk of the cAMP and

cGMP pathways (Zaccolo & Movsesian 2007). All the PDEs

consist of a modular architecture, with variable regulatory

domains located at the N-terminus and a highly conserved

catalytic region at the C-terminus (Conti & Beavo 2007).

PDE4A5 The cAMP-specific PDE4 group forms the largest

PDE subfamily, which is the main enzyme responsible for

cAMP degradation (Lugnier 2006). PDE4s can be distin-

guished from other PDE subfamilies by sequence identity in

the catalytic region and by the presence of specific regions,

located at the N-terminal portion of the proteins, called

upstream conserved regions 1 and 2 (UCR1 and UCR2;

Bolger et al. 2003, de Oliveira & Smolenski 2009). At least 35

splice variants are encoded by four genes (PDE4A–D;

Lugnier 2006). Of these, the highly conserved PDE4A4/5

isoform (PDE4A5 is the rat homologue of the human

PDE4A4) is characterised by an extended N-terminal region

involved in subcellular targeting (Bolger et al. 2003).

PDE4A4/5 is expressed in a wide variety of tissues, including

the pituitary (Mackenzie et al. 2008, Lennox et al. 2011), and

both membrane and cytosolic localisation has been detected

(Huston et al. 2000). PDE4A5 was demonstrated to interact

with the SH3 domains of SRC family tyrosyl kinases

(O’Connell et al. 1996, Beard et al. 2002), with AKAP3

(Bajpai et al. 2006) and with AIP (Bolger et al. 2003). AIP was

initially identified as a direct binding partner of PDE4A5 by a

Y2H screening of a rat brain cDNA library. The interaction

was subsequently confirmed by a GST pull-down assay and




was also demonstrated in mammalian cells. In addition, the

ability of AIP to bind PDE4A5 was found to be unaffected by

the concomitant binding of LYN, a member of the tyrosyl

kinase family.

The interaction was proved to be highly specific, because

other immunophilins or TPR-containing proteins such as the

AIP homologue AIPL1, FKBP51 and FKBP52 were unable

to bind PDE4A5 and do not involve other PDE4 isoforms.

The domains mediating the interaction were mapped in the

TPR region of AIP and in both the unique N-terminal

region (amino acids 11–42) and the UCR2 domain (only the

PDE4-conserved EELD motif) of PDE4A5. The binding of

AIP to PDE4A5 was demonstrated to lead to three distinct

functional consequences. First is a reversible, dose-dependent

inhibition of PDE4A5 catalytic activity by w60%, which is

directly mediated by the TPR domain of AIP. A second

outcome, also leading to a decreased enzymatic activity, is the

attenuation of PDE4A5 phosphorylation by PKA. The third

consequence is an increased sensitivity of PDE4A5 to

rolipram, the PDE4-specific inhibitor.

The impact of several missense and nonsense AIP mutations

on PDE4A5 binding was studied by a Y2H b-galactosidase

assay (Bolger et al. 2003, Leontiou et al. 2008, Igreja et al.

2010). All four naturally occurring truncating mutations

investigated (p.R81X, p.Q164X, p.Q217X and p.R304X)

completely abolish the interaction (Leontiou et al. 2008, Igreja

et al. 2010). The 12 missense mutations analysed were selected

based on the evolutionarily conserved structurally critical

amino acids located within the consensus TPR domain (Das

et al. 1998, Russell et al. 1999, Scheufler et al. 2000) (p.N236A,

p.K266A and p.R271A) (Bolger et al. 2003) or because they

were described in patients or controls (p.R16H, p.V49M,

p.K103R, p.C238Y, p.K241E, p.I257V, p.R271W, p.A299V

and p.R304Q) (Leontiou et al. 2008, Igreja et al. 2010).

Regarding p.N236A, p.K266A and p.R271A, it was shown

that only the latter significantly attenuates the interaction of

AIP with PDE4A5 and also its ability to inhibit PDE4A5,

whereas the others exhibit normal binding and do not

compromise the inhibitory capacity of AIP (Bolger et al.

2003). The other nine missense variants investigated can be

divided into two groups: the first group comprises mutations

with b-galactosidase assay activity values more than fivefold

different from WT (p.K103R, p.C238Y, p.K241E and

p.R271W), suggesting that they lead to a complete loss of

PDE4A5–AIP binding (Leontiou et al. 2008, Igreja et al.

2010), whereas the other group has normal (p.V49M) or less

than threefold different b-galactosidase assay activity values

from WT (p.R16H, p.I257V, p.A299V and p.R304Q). The

low impact of the p.R16H variant on PDE4A5 binding

(Igreja et al. 2010), together with its normal interaction with

the RET protein (described in RET section; Georgitsi et al.

2007), suggest that if this is a pathogenic variant and not a

rare polymorphism, then based on the in vitro studies,

a PDE4A5- and RET-independent mechanism may take

place. Notably, the p.R304Q variant has very strong clinical

data suggesting that it is a disease-causing variant, but the


in vitro b-galactosidase assay was not showing strong

abnormality (Igreja et al. 2010). Clearly, further data are

needed to understand whether PDE4A5 binding correlates

with the tumorigenic properties of AIP.

Combining together the results from these and other

binding studies (Bolger et al. 2003, Laenger et al. 2009, Igreja

et al. 2010), the essential role played by the R271 residue in

mediating the binding of AIP with hsp90 and PDE4A5

emerges. This amino acid is likely to participate in an

electrostatic interaction with the EEVD and EELD motifs of

hsp90 and PDE4A5 respectively (Bolger et al. 2003), and its

mutation completely disrupts these interactions. It is also

interesting to note that the p.R271W human mutation affects

a CpG site and is the second most common mutational

hotspot described in the AIP gene (Igreja et al. 2010).

PDE2A3 Three isoforms of PDE2A (PDE2A1, PDE2A2

and PDE2A3), generated from a single gene by alternative

splicing, have been cloned from several different species. These

three isoforms are identical except for the N-terminal regions,

which are responsible for their different subcellular local-

isation. The human variant (PDE2A3) encodes a membrane-

associated protein of 941 amino acids. PDE2A functions as a

homodimer and each monomer is formed by an N-terminal

domain, two tandem GAF domains (GAF-A and -B) and a

catalytic C-terminal domain. PDE2A is able to hydrolyse both

cAMP and cGMP, but in the presence of cGMP, which binds

to the allosteric GAF-B domain, the enzyme is activated and

increases its affinity for cAMP, resulting in a greater

hydrolysing capacity for cAMP than for cGMP (Bender &

Beavo 2006). This enzyme thus contributes to the crosstalk

between these two second messenger pathways (Zaccolo &

Movsesian 2007). PDE2A is strongly expressed in the brain

with a moderate presence in peripheral tissues such as the

adrenal gland and heart and skeletal muscle (Rosman et al.

1997, Sadhu et al. 1999, Stephenson et al. 2009, Lin et al.

2010). In addition, PDE2A expression has been observed in rat

(Velardez et al. 2000, Stephenson et al. 2009) and human

pituitary (Lennox et al. 2011).

de Oliveira et al. (2007) identified an interaction between

the human PDE2A and the AIP by a Y2H screening of a

human brain cDNA library. The interaction was subsequently

confirmed by GST pull-down and co-IP experiments both in

cell lines and in brain tissue lysates. The two proteins were

found to colocalise in the cytosol, with the occasional

involvement of the plasma membrane. The regions that

mediate the interaction were mapped in the GAF-B domain

of PDE2A and the C-terminal half (amino acids 170–330) of

AIP. In addition, preliminary results suggested that AIP was

able to interact with PDE2A and hsp90 at the same time.

Different from what was previously shown for PDE4A5, the

enzymatic activity of PDE2A was unaffected by AIP binding.

PDE2A, due to its binding to AIP, is located in the vicinity of

AhR and is able to lower the local cAMP concentration in the

compartment where the AhR complex is located, therefore

the TCDD- and especially the forskolin-induced nuclear




translocation of AhR is 40% (with TCDD) or 55% (with

forskolin) lower in PDE2A-transfected cells compared with

control cells. Furthermore, this inhibition was demonstrated

to correlate with a reduction of the AhR function, as reported

by a reporter gene assay.

At present, it is unknown if both PDE4A5 and PDE2A

can simultaneously associate via AIP to the AhR complex

(de Oliveira & Smolenski 2009).

Nuclear receptors

Several studies demonstrated the involvement of AIP in

various nuclear receptor signalling pathways.

Oestrogen receptor a ERa and ERb mediate the

biological effects of oestrogens both in reproductive and in

nonreproductive organs in both sexes. After ligand binding,

the ERs sequentially dimerise, translocate into the nucleus,

associate with several coregulator proteins, and regulate the

transcriptional activity of oestrogen target genes (Nilsson &

Gustafsson 2011). ERa and ERb display distinct or even

opposite effects: in tumour cells, ERa was shown to promote

cell proliferation (Zeng et al. 2008), whereas ERb stimulates

apoptosis (Cheng et al. 2004). The physiological actions of

oestrogens are thus the result of a balance between ERa and

ERb signalling (Heldring et al. 2007).

It has recently been shown that AIP is involved in ERatranscriptional activation by interacting with the co-activator

TIF-2 (Pongratz et al. 2009), which is structurally related to

members of the bHLH–PAS family (Voegel et al. 1998). In

particular, AIP negatively regulates the protein levels of TIF-2

both in the presence and in the absence of ligand, thus

exerting a negative effect on ERa transcriptional activity. We

could hypothesise that AIP may have a role in preventing

ERa-dependent tumour growth.

Glucocorticoid receptor The GR is a member of the

nuclear receptor superfamily. Like the AhR, it has been

demonstrated to exist in a multiprotein heterocomplex

containing two molecules of hsp90 and other co-chaperone

proteins, such as p23, PP5 and FKBP52 (Grad & Picard

2007). Moreover, the GR signalling pathway shares some

mechanistic similarities with the AhR pathway. Both the

receptors reside in the cytoplasm in the absence of the

respective ligands and, on their binding, they undergo

conformational changes, which lead to nuclear translocation.

Inside the nucleus, the GR homodimerises and the dimer

binds to the recognised enhancer elements, activating the

transcription of the target genes (Heitzer et al. 2007). The

similar mechanism of action with the AhR and especially the

presence of TPR-containing proteins as GR regulators

prompted different groups to test whether AIP is part of the

GR complex.

The first studies by Carver et al. (1998), conducted by a

Y2H screening (Carver & Bradfield 1997) and by co-IP

experiments in yeast, demonstrated that AIP does not interact


with the GR complex. However, subsequently, co-IP assays

in a mammalian cell line showed that this interaction occurs

via hsp90 (Laenger et al. 2009). The effect of AIP on the GR

signalling is inhibitory due to a delayed nuclear accumulation

of GR after ligand binding, with subsequent decrease of

GR’s transcriptional activity (Laenger et al. 2009, Schulke

et al. 2010). A similar situation has occurred with FKBP51,

which was found not to be associated with GR in yeast but

was associated in mammalian cells. A possible explanation

could be the lack of other TPR domain protein partners of

the receptor resulting in suboptimal activity in yeast (Laenger

et al. 2009).

Apart from ER and GR, some evidences suggest the

potential of AIP to interact with other steroid hormone

receptors. In particular, AIP has recently been shown to

strongly inhibit the transcriptional activity of the receptors for

progesterone (PR) and androgen (AR) (Schulke et al. 2010).

Peroxisome proliferator-activated receptor a The

peroxisome proliferator-activated receptor a (PPARa,

MIM# 170998) is a soluble transcription factor belonging,

as the GR, to the nuclear receptor superfamily. PPARa can

be activated by different lipophilic compounds, and, in turn, it

associates with the retinoid X receptor a (RXRa). This

heterodimer activates the transcription of several genes

encoding enzymes involved in the lipid and lipoprotein

metabolism (Yoon 2009).

By co-IP experiments, using an antibody highly specific for

the PPARa isoform and therefore not binding to the other

PPARs subtypes PPARb and PPARg, the mouse PPARawas

found to form a complex with AIP and hsp90 in the liver

cytosol. However, as PPARa is predominantly nuclear, the

authors hypothesised that this complex could exist as well in

the nucleus (Sumanasekera et al. 2003). Similar to the

inhibition exerted on the transcriptional activity of the

HBV X protein and GR (Kuzhandaivelu et al. 1996, Laenger

et al. 2009), AIP was found to repress PPARa activity when

overexpressed (Sumanasekera et al. 2003). However, the

normally low expression levels of AIP in the liver, in contrast

to the high PPARa expression, suggest that the inhibitory

effect of AIP might be very weak or not explicated at all in

physiological conditions.

Thyroid hormone receptors b1 Thyroid hormone

receptors (TRs) mediate the genomic actions of the thyroid

hormone triiodothyronine (T3). TRs are nuclear receptors

derived from two genes, THRA and THRB. The THRB

(MIM# 190160) gene encodes three isoforms, b1, b2 and b3

(Cheng et al. 2010). TRb1 is involved in the negative

feedback of T3 on TRH production in the paraventricular

nucleus (PVN) but also mediates the T3-independent

activation of TRH transcription. As the mechanism by

which TRb1 exerts this activating role was unknown,

Froidevaux et al. (2006) decided to look for TRb1-interacting

proteins. Using a Y2H assay to screen a mouse PVN cDNA,

AIP was identified as a new TRb1 partner. AIP and TRb1




colocalise in the same neurons in the PVN, thus giving to this

interaction a functional significance in the regulation of TRH

activation. The AIP–TRb1 interaction was demonstrated to

be very specific both in yeast and in a mammalian cell line, as

no other TR isoforms interacted with AIP. However, the

strength of the complex in yeast was T3 dependent, whereas

in mammals the interaction (although weaker) was still

happening in the absence of T3. AIP binds to TRb1 via

the TPR domain, because its deletion causes the loss of the

interaction. AIP, however, does not change the subcellular

localisation of TRb1, regardless of the presence of the ligand

T3. An in vitro small inhibitory RNA (siRNA) experiment

demonstrated that the stability of the TRb1 receptor was

compromised by AIP knockdown. AIP binding to TRb1 is

independent of the presence of T3 in mammals. In vivo

siRNA studies show that AIP is indispensable for the

T3-independent TRb1 activation of TRH transcription but

is not necessary for the T3-dependent repression.

Transmembrane receptors

Rearranged during transfection The rearranged during

transfection (RET) (MIM# 164761) proto-oncogene encodes

a transmembrane tyrosine kinase receptor involved in the

development, maturation and survival-controlling functions

of epithelial, neuronal and several neuroendocrine cells (Lai

et al. 2007). RET has been shown to be expressed in two major

splicing isoforms that differ at the C-terminal end: a long

isoform of 1114 amino acids, termed RET51, and a short

isoform, 1072 amino acids long, named RET9 (Tahira et al.

1990). Structurally, RET can be divided into three domains: a

large extracellular domain that includes a cadherin-like and a

cysteine-rich region, a transmembrane domain, and an

intracellular tyrosine kinase domain (Lai et al. 2007). In the

presence of the ligand RET activates various signal transduc-

tion pathways that ultimately promote survival, growth and

extension/migration of cells, whereas in its absence, it releases

an intracellular fragment that induces apoptosis (Canibano

et al. 2007). RET activating mutations have been associated

with the following disorders: MEN2A (MIM# 171400)

and MEN2B (MIM# 162300), familial medullary thyroid

carcinoma (MIM# 155240) and RET inactivation mutations

with Hirschsprung disease (MIM# 142623). None of these

diseases are associated with pituitary adenomas and no RET

mutations have been identified in FIPA families (Lai et al.

2007, Vargiolu et al. 2009, Heliovaara et al. 2010).

In order to identify novel signalling pathways induced by

RET51, Vargiolu et al. (2009) used the new split-ubiquitin

Y2H assay (Dirnberger et al. 2008) to screen a human fetal

cDNA library. Among the ten clones identified, one

corresponded to AIP. Interestingly, another clone was the

AIP-related immunophilin FKBP52 (Fusco et al. 2010). The

AIP–RET interaction was subsequently validated by a co-IP

assay and was also shown to occur with both RET isoforms.

Moreover, the AIP–RET complex was demonstrated to be

present in cells endogenously expressing both proteins and


also in vivo in the rat pituitary gland tissue. The region of RET

involved in the interaction was mapped between residues 707

and 999, a region common to both isoforms located within

the intracellular proapoptotic fragment. The interaction was

also found to be retained even if the kinase activity of RET

was suppressed. In addition, the impact of six AIP missense

mutations (p.R16H, p.K241E, p.R271W, p.E293G, p.A299V

and p.R304Q) on RET binding was tested by co-IP

experiments. None of them was found to affect the

interaction of AIP with the two RET isoforms.

RETwas also demonstrated to interfere with AIP–survivin

interaction (Vargiolu et al. 2009), an effect that will be

discussed in the Survivin section.

Epidermal growth factor receptor The epidermal

growth factor receptor (EGFR, MIM# 131550) is a

transmembrane glycoprotein, which is required for normal

cellular proliferation, survival, adhesion, migration and

differentiation (Harari et al. 2007). EGFR interacts with a

wide range of proteins, among which AIP was reported

(Deribe et al. 2009). AIP was identified in a large-scale

MYTH screen, a split-ubiquitin-based membrane Y2H assay,

which allows the systematic analysis of full-length membrane

protein interactions in a cellular environment (Snider et al.

2010). However, this interaction was not subjected to further

confirmations employing different techniques and thus, given

the high rate of false positives in Y2H analysis (Fields 2005), it

cannot be considered certain. It would be important to

confirm this interaction in the pituitary because EGFR

inhibition has been shown to control pituitary tumour

growth and hormone secretion (Vlotides et al. 2008).

G proteins

G proteins are heterotrimeric GTP-binding proteins formed

by a, b and g subunits (Ga, Gb and Gg), which mediates

receptor-stimulated signalling pathways. Ga subunits are

typically divided into four families, Gas, Gai/Gao, Gaq/Ga11

and Ga12/Ga13. Ga13, similar to AIP, is ubiquitously

expressed and is an essential gene, because its deletion is

embryonic lethal in mice (Worzfeld et al. 2008).

Using Y2H screening of a mouse fetal brain cDNA, Nakata

et al. (2009), looking for new Ga13-interacting proteins,

found that AIP is a binding partner of Ga13. This interaction

was confirmed in vitro by a GST pull-down assay and was

shown to involve the whole AIP protein sequence. It was

also determined that the interaction is independent of the

GTP/GDP binding status of the a subunit and that it does not

involve the b and g subunits. The other three types of asubunit were also tested for their ability to interact with AIP,

and it was found that the Gaq subunit also binds AIP, although

weaker than Ga13, whereas Gas and Gai do not bind AIP. It is

interesting to note that another TPR protein, PP5, also binds

Ga12/Ga13 (Yamaguchi et al. 2002).

Ga13 activation was then demonstrated to disturb the

AIP–AhR interaction through the destabilisation of the AhR




protein via the ubiquitin–proteasome pathway and was also

shown to inhibit the ligand-mediated transcriptional acti-

vation of the AhR independently from RhoA binding (RhoA

is a small GTP-binding protein that is activated after the

interaction with Ga13). Gaq, despite its less tight interaction

with AIP, was also found to exert a strong inhibitory effect on

the AhR (Nakata et al. 2009). The downstream signalling

pathways regulated by Ga13, apart from the activation of

RhoA, are not well characterised (Worzfeld et al. 2008).

Because the Ga13-mediated inhibition of AhR explicates in a

RhoA-independent manner, what happens to Ga13 signalling

is at present unknown. However, it is interesting to note that

there are some evidences reporting the involvement of the

Ga13 pathway in the regulation of cAMP responses (Jiang

et al. 2007, 2008). Specifically, the synergistic regulation of

cAMP synthesis by the Gas and Ga13 pathways was shown to

be mediated by a specific isoform of adenylyl cyclase, termed

AC7 (Jiang et al. 2008).

AIP

Mitochondrial protein

Phosphodiesterase

Kinase

Cytoskeletal protein

Chaperone

Viral protein

Gα13 Gαq

PPARα

TRβ1

RET

Survivin

TOMM20

PDE2A3PDE4A5

EGFR

TNNI3K

Actin

hsc70

hsp90

GR

p23AhR

EBNA-3

HBVX

ERα

TIF-2

Co-chaperone

G protein

Nuclear receptor

Transmembrane receptor

Inhibitor of apoptosis

Co-activator

Figure 5 AIP interactome. The protein–protein interaction networkwas rendered with NAViGaTOR 2.1.13 (http://ophid.utoronto.ca/navigator; Brown et al. 2009). Nodes correspond to proteins andlines to physical protein–protein interactions. Solid lines signifyconfirmed interactions, whereas dashed lines represent uncertainassociations. Node colours discriminate between different classesof proteins.


The AhR dissociation from AIP induced by Ga13 was

shown to cause the translocation of the AhR into the nucleus

in a ligand-independent manner. However, the AhR that

moves into the nucleus in this way does not form an active

complex with ARNT, as also seen for the cAMP-mediated

nuclear translocation of the AhR (Oesch-Bartlomowicz et al.

2005). This divergence suggests that the AhR adopts a unique

structure in the cytoplasm and that AhR does not undergo the

same conformational change when moving to the nucleus

with every nuclear transport inducer (such as TCDD, Ga13

or cAMP).

TOMM20 and mitochondrial preproteins

TOMM20 is an import receptor that, along with TOMM70

and other proteins, is part of the TOMM protein complex.

The TOMM protein complex is involved in the recognition

and translocation of mitochondrial preproteins synthesised

in the cytoplasm. To be able to cross the mitochondrial

membranes, the preproteins are maintained in the cytosol in

an unfolded translocation-competent conformation by

different molecular chaperones (Baker et al. 2007). Among

them, hsp90 and hsc70 have been shown to mediate the

import of TOMM70-dependent preproteins (Young et al.

2003), and AIP was demonstrated to interact with TOMM20

(Yano et al. 2003). In humans, TOMM20 can be structurally

subdivided into five regions: a transmembrane segment at the

N-terminus, a linker segment, a TPR motif, a Q-rich region

and a conserved COOH-terminal acidic segment (Abe et al.

2000). This latter region is not required for preprotein

binding but is specifically involved in an electrostatic

interaction with the TPR motifs of AIP (Yano et al. 2003).

In particular, an essential role has been found to be played by

the very last five amino acids of TOMM20 (EDDVE), a

segment similar to those present in PDE4A5 (EELD, involved

in AIP binding) (Bolger et al. 2003) and hsp90 and hsc70

(EEVD, involved in the interaction with various TPRs)

(Smith 2004).

In the same study (Yano et al. 2003), AIP was also shown

to interact with several mitochondrial preproteins, for

example preornithine transcarbamylase. Different from the

AIP–TOMM20 interaction, both the PPIase-like and the

TPR regions of AIP are required to bind the internal import

signals of the preproteins. AIP was then demonstrated to

maintain the loosely folded state of preproteins and to

promote their transfer into mitochondria. This finding was

confirmed by the demonstration that AIP forms a ternary

complex with TOMM20 and the preprotein and by the fact

that AIP interacts with preproteins less strongly than with

TOMM20. Altogether, these results suggest the following

scenario: mitochondrial preproteins may form a large

complex in the cytosol with hsc70 and AIP, with both

proteins contributing to maintain their unfolded confor-

mation; once the complex has reached the outer membrane

of the mitochondria, AIP binds to TOMM20 and promotes

the transfer of the preprotein to the import receptor.



http://ophid.utoronto.ca/navigator

http://ophid.utoronto.ca/navigator

Table 1 Interacting partners of aryl hydrocarbon receptor-interacting protein (AIP). The techniques used to identify the various interactions,the functions of the different AIP partners, the effect of AIP on the partner’s activity/stability and the organisms/cell types where the interactionshave been proved are reported

Partner Y2H co-IP Pull-down OtherConfirmedinteraction Function

AIP effect onthe partner’sactivity/stability

Organism and/or cell type(s)

HBV X # # Y Transcriptionalactivator

Y Human lymphoma,HeLa cells

EBNA-3 # # Y Immortalization andtransformation ofB-cells

NE EBV-immortalizedlymphoblastoidcells, human lym-phocytes and fetalbrain

AhR # # # # Y Adaptive and toxicresponses, develop-ment

[Y HeLa, Hepa1c1c7,COS-1, B-cells

Hsp90 # # # Y Protein folding, mito-chondrial proteinimport

NE HeLa, HEK, COS-1,COS-7, bacterialcells

Hsc70 # Y Protein folding, mito-chondrial proteinimport, disassemblyof clathrin-coatedvesicles

NE HeLa cells

Actin # N Cytoskeletalcomponent

NE COS-7 cells

PDE4A5 # # # Y cAMP degradation Y Rat brain, COS-7 cellsPDE2A3 # # # Y cAMP and cGMP

degradationZ Human brain, COS-1,

HeLa cellsTIF-2 # # Y Activates ERa Y HC11 cellsPPARa # # Y Regulation of energy

homeostasisY Mouse liver

TRb1 # # Y Activation of TRHtranscription

[ Mouse PVN of thehypothalamus

RET # # Y Development, matu-ration, survival

NE Human fetal brain, ratpituitary, neuro-blastoma andHEK293 cells

EGFR # N Cellular proliferation,survival, adhesion,migration, differen-tiation

NE Human fetal brain

Ga13 # # Y Mediates receptor-stimulated signal-ling pathways

NE HEK293T,Hepa1c1c7, COS-7cells

Gaq # Y Mediates receptor-stimulated signal-ling pathways

NE HEK293T, Hepa1c1c7cells

TOMM20 # # Y Mitochondrial importreceptor

NE Human fetal liver,COS-7, HeLa cells

Survivin # # # Y Suppression ofapoptosis

[ HeLa, MCF-7, Rajicells

TNNI3K # N Promotes cardiomyo-genesis, enhancescardiac per-formance, protectsthe myocardiumfrom ischemicinjury

NE Human heart

Y, yes; N, no; NE, not evaluated; [, increase; Y, decrease; Z, no effect.


www.endocrinology-journals.org Journal of Endocrinology (2011) 210, 137–155



Survivin

Survivin is a member of the inhibitor of apoptosis (IAP) gene

family, which includes evolutionarily conserved members

that suppress apoptosis by preventing the maturation and/or

the proteolytic activity of its effector enzymes, the caspases

(Wei et al. 2008). Survivin, apart from inhibiting apoptosis,

is also implicated in other essential cellular functions like

the control of cell division and the stress response (Fortugno

et al. 2003, Dohi et al. 2004, Yang et al. 2004). For instance,

during harmful environmental stimuli, a significant release

of survivin takes place from the mitochondria to the cytosol

(Dohi et al. 2004) and survivin form complexes with hsp90

(Fortugno et al. 2003). Structurally, survivin is a 142 amino

acid-long protein that exists as a functional homodimer.

The dimerisation is mediated by the N-terminal baculovirus

IAP repeat (BIR) domain, which is also involved in the

interaction with other proteins, i.e. the caspases and hsp90

(Chantalat et al. 2000, Verdecia et al. 2000, Fortugno et al.

2003). The survivin–AIP interaction, discovered in a

proteomics screening by Kang & Altieri (2006) is instead

mediated by the C-terminal a-helical coiled-coil region,

with a key role played by the last residue of the protein. The

AIP region involved in the binding was mapped between

residues 170 and 330, thus comprising the three TPR motifs.

The interaction was demonstrated to be direct and to happen

in pull-down and co-IP experiments. The association of

AIP with survivin was shown to stabilise survivin protein

levels independently from hsp90 binding (Fortugno et al.

2003). Loss of both proteins leads to proteasomal degradation

of survivin, resulting in enhanced apoptosis and, only for

hsp90, also to cell cycle arrest. The reported association of

AIP with TOMM20 (Yano et al. 2003) leaves room to

speculate that AIP could stabilise survivin in the cytoplasm

and facilitate its displacement into the mitochondria pool,

increasing the reserves of survivin in this compartment

readily for subsequent events that involve apoptosis (Kang &

Altieri 2006). This hypothesis has recently been experimen-

tally confirmed by the same group (Kang et al. 2011).

However, because several evidences suggest that AIP acts

as a tumour suppressor gene (Leontiou et al. 2008), it is

not clear why it stabilises survivin, thereby elevating the

cellular anti-apoptotic threshold. To further increase the

level of complexity of the system, the RET protein

abolishes the binding of AIP to survivin (Vargiolu et al.

2009), probably promoting apoptosis, a role already docu-

mented for RET (Bordeaux et al. 2000, Canibano et al.

2007). It is thus conceivable that when AIP and RET are

co-expressed in the same tissue, RET acts to counteract

the AIP-mediated stabilisation of survivin, in order to lower

the anti-apoptotic threshold.

TNNI3K

Zhao et al. (2003) cloned a new cardiac-specific kinase gene

named TNNI3K (cardiac troponin I-interacting kinase),


belonging to the MAPKK family. In this study, TNNI3K

was found to interact with several proteins including AIP, by a

Y2H screening. The interaction with AIP was not further

investigated with other techniques. In the light of the recent

findings that AIP is essential in cardiac development (Lin et al.

2007) and TNNI3K plays an important role in the cardiac

system (Lai et al. 2008), it will be of great interest to study

more deeply this interaction.

Conclusions

Twenty interactions have been described for AIP. Fourteen

proteins have been shown to directly interactwith AIP: twoviral

proteins (HBV X and EBNA-3), two chaperone proteins

(hsp90 and hsc70), two PDEs (PDE4A5 and PDE2A3), three

nuclear (AhR, PPARa and TRb1) and a transmembrane

(RET) receptors, two G proteins (Ga13 and Gaq), an IAP

(survivin) and a mitochondrial import receptor (TOMM20);

three non-confirmed interactions with a cytoskeletal protein

(actin), a growth factor receptor (EGFR) and a cardiac-specific

kinase (TNNI3K) have also been described. The association of

AIP with the GR and the co-chaperone protein p23 was proved

to be mediated by hsp90, whereas the AIP–ERa interaction

involves the ERa co-activator TIF-2 (Fig. 5 and Table 1).

Although the majority of studies regarding AIP have so

far focused mainly on the AhR pathway, lots of interactions

with functionally different proteins have also been reported,

indicating that AIP is involved in various cellular pathways.

The role of AIP in all these pathways needs to be further

investigated as well as the possible links between them,

as it is beginning to unravel, for instance, between the

AhR–cAMP–PDE and AhR–EGFR pathways (Haarmann-

Stemmann et al. 2009, Oesch-Bartlomowicz & Oesch 2009,

de Oliveira & Smolenski 2009).

Currently, it is uncertain which of the AIP interactions play

a role in pituitary tumorigenesis. The AhR–cAMP–PDE

pathway seems to be a very attractive candidate as AhR

activation has well-known tumorigenic effects (Dietrich &

Kaina 2010) and PDEs are involved in the tight control of

cellular cAMP levels (Zaccolo & Movsesian 2007), which are

known to be involved in Gas mutation- and CNC-related

somatotroph tumorigenesis (Boikos & Stratakis 2007).

Specially, the loss of PDE2A3 is postulated to lead to

increased cAMP levels, which could result in hypertrophy and

hyperplasia of the pituitary, possibly involving down-

regulation of ARNT (Heliovaara et al. 2009). Also the

interaction between RET and AIP might have a role in

pituitary adenoma tumorigenesis as the loss of RET results in

somatotroph hyperplasia (Canibano et al. 2007). However,

neither AIP nor RET mutations were shown to affect their

interaction in vitro (Vargiolu et al. 2009). Further studies are

thus indispensable to identify the exact mechanism by which

AIP causes pituitary tumorigenesis as this might ultimately

lead to the design of novel strategies to cure the disease.




Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/

JOE-11-0054.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived

as prejudicing the impartiality of the research reported.

Funding

This research did not receive any specific grant from any funding agency in the

public, commercial or not-for-profit sector.

References

Abe Y, Shodai T, Muto T, Mihara K, Torii H, Nishikawa S, Endo T &

Kohda D 2000 Structural basis of presequence recognition by the

mitochondrial protein import receptor Tom20. Cell 100 551–560.

(doi:10.1016/S0092-8674(00)80691-1)

Alberts B, Johnson A, Lewis J, Raff M, Roberts K & Walter P 2002

The molecular mechanisms of vesicular transport and the maintenance

of compartmental diversity. In Molecular Biology of the Cell, 4th edition.

Garland Science, New York.

Antonsson C, Whitelaw ML, McGuire J, Gustafsson JA & Poellinger L 1995

Distinct roles of the molecular chaperone hsp90 in modulating dioxin

receptor function via the basic helix–loop–helix and PAS domains.

Molecular and Cellular Biology 15 756–765.

Bajpai M, Fiedler SE, Huang Z, Vijayaraghavan S, Olson GE, Livera G,

Conti M & Carr DW 2006 AKAP3 selectively binds PDE4A isoforms in

bovine spermatozoa. Biology of Reproduction 74 109–118. (doi:10.1095/

biolreprod.105.043588)

Baker MJ, Frazier AE, Gulbis JM & Ryan MT 2007 Mitochondrial protein-

import machinery: correlating structure with function. Trends in Cell Biology

17 456–464. (doi:10.1016/j.tcb.2007.07.010)

Beard MB, Huston E, Campbell L, Gall I, McPhee I, Yarwood S, Scotland G

& Houslay MD 2002 In addition to the SH3 binding region, multiple

regions within the N-terminal noncatalytic portion of the cAMP-specific

phosphodiesterase, PDE4A5, contribute to its intracellular targeting.

Cellular Signalling 14 453–465. (doi:10.1016/S0898-6568(01)00264-9)

Beischlag TV, Luis MJ, Hollingshead BD & Perdew GH 2008 The aryl

hydrocarbon receptor complex and the control of gene expression. Critical

Reviews in Eukaryotic Gene Expression 18 207–250.

Bell DR & Poland A 2000 Binding of aryl hydrocarbon receptor (AhR) to

AhR-interacting protein. The role of hsp90. Journal of Biological Chemistry

275 36407–36414. (doi:10.1074/jbc.M004236200)

Bender AT & Beavo JA 2006 Cyclic nucleotide phosphodiesterases: molecular

regulation to clinical use. Pharmacological Reviews 58 488–520. (doi:10.

1124/pr.58.3.5)

Benlian P, Giraud S, Lahlou N, Roger M, Blin C, Holler C, Lenoir G,

Sallandre J, Calender A & Turpin G 1995 Familial acromegaly: a specific

clinical entity – further evidence from the genetic study of a three-

generation family. European Journal of Endocrinology 133 451–456. (doi:10.

1530/eje.0.1330451)

Berg P & Pongratz I 2002 Two parallel pathways mediate cytoplasmic

localization of the dioxin (aryl hydrocarbon) receptor. Journal of Biological

Chemistry 277 32310–32319. (doi:10.1074/jbc.M203351200)

Blatch GL, Cox MB & Smith DF 2006 Functions of the Hsp90-binding FKBP

immunophilins. In Networking of Chaperones by Co-Chaperones, pp 13–23.

Landes Bioscience, Austin, Texas. (doi:10.1007/978-0-387-49310-7_2)

Blom E, van de Vrugt HJ, de VY, de Winter JP, Arwert F & Joenje H 2004

Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

DNA Repair 3 77–84. (doi:10.1016/j.dnarep.2003.09.007)


Boikos SA & Stratakis CA 2007 Molecular genetics of the cAMP-dependent

protein kinase pathway and of sporadic pituitary tumorigenesis. Human

Molecular Genetics 16 R80–R87. (doi:10.1093/hmg/ddm019)

Bolger GB, Peden AH, Steele MR, MacKenzie C, McEwan DG, Wallace DA,

Huston E, Baillie GS & Houslay MD 2003 Attenuation of the activity of the

cAMP-specific phosphodiesterase PDE4A5 by interaction with the

immunophilin XAP2. Journal of Biological Chemistry 278 33351–33363.

(doi:10.1074/jbc.M303269200)

Bordeaux MC, Forcet C, Granger L, Corset V, Bidaud C, Billaud M,

Bredesen DE, Edery P & Mehlen P 2000 The RET proto-oncogene

induces apoptosis: a novel mechanism for Hirschsprung disease. EMBO

Journal 19 4056–4063. (doi:10.1093/emboj/19.15.4056)

Brown KR, Otasek D, Ali M, McGuffin MJ, Xie W, Devani B, Toch IL &

Jurisica I 2009 NAViGaTOR: network analysis, visualization and graphing

Toronto. Bioinformatics 25 3327–3329. (doi:10.1093/bioinformatics/

btp595)

Cain JW, Miljic D, Popovic V & Korbonits M 2010 Role of the aryl

hydrocarbon receptor-interacting protein in familial isolated pituitary

adenoma. Expert Review of Endocrinology and Metabolism 5 681–695.

(doi:10.1586/eem.10.42)

Canibano C, Rodriguez NL, Saez C, Tovar S, Garcia-Lavandeira M,

Borrello MG, Vidal A, Costantini F, Japon M, Dieguez C et al. 2007

The dependence receptor Ret induces apoptosis in somatotrophs

through a Pit-1/p53 pathway, preventing tumor growth. EMBO Journal

26 2015–2028. (doi:10.1038/sj.emboj.7601636)

Carver LA & Bradfield CA 1997 Ligand-dependent interaction of the aryl

hydrocarbon receptor with a novel immunophilin homolog in vivo.

Journal of Biological Chemistry 272 11452–11456. (doi:10.1074/jbc.272.17.

11452)

Carver LA, LaPres JJ, Jain S, Dunham EE & Bradfield CA 1998

Characterization of the Ah receptor-associated protein, ARA9. Journal of

Biological Chemistry 273 33580–33587. (doi:10.1074/jbc.273.50.33580)

Cazabat L, Libe R, Perlemoine K, Rene-Corail F, Burnichon N, Gimenez-

Roqueplo AP, Dupasquier-Fediaevsky L, Bertagna X, Clauser E, Chanson P

et al. 2007 Germline inactivating mutations of the aryl hydrocarbon receptor-

interactingproteingene in a large cohort of sporadic acromegaly: mutations are

found in a subset of young patients with macroadenomas. European Journal of

Endocrinology 157 1–8. (doi:10.1530/EJE-07-0181)

Chahal HS, Chapple JP, Frohman LA, Grossman AB & Korbonits M 2010

Clinical, genetic and molecular characterization of patients with familial

isolated pituitary adenomas (FIPA). Trends in Endocrinology and Metabolism 21

419–427. (doi:10.1016/j.tem.2010.02.007)

Chahal HS, Stals K, Unterlander M, Balding DJ, Thomas MG, Kumar AV,

Besser GM, Atkinson AB, Morrison PJ, Howlett TA et al. 2011 AIP

mutation in pituitary adenomas in the 18th century and today. New England

Journal of Medicine 364 43–50. (doi:10.1056/NEJMoa1008020)

Chantalat L, Skoufias DA, Kleman JP, Jung B, Dideberg O & Margolis RL

2000 Crystal structure of human survivin reveals a bow tie-shaped dimer

with two unusual alpha-helical extensions. Molecular Cell 6 183–189.

(doi:10.1016/S1097-2765(05)00020-1)

Chen HS & Perdew GH 1994 Subunit composition of the heteromeric

cytosolic aryl hydrocarbon receptor complex. Journal of Biological Chemistry

269 27554–27558.

Chen MS, Silverstein AM, Pratt WB & Chinkers M 1996 The

tetratricopeptide repeat domain of protein phosphatase 5 mediates binding

to glucocorticoid receptor heterocomplexes and acts as a dominant negative

mutant. Journal of Biological Chemistry 271 32315–32320. (doi:10.1074/jbc.

271.50.32315)

Cheng J, Lee EJ, Madison LD & Lazennec G 2004 Expression of estrogen

receptor beta in prostate carcinoma cells inhibits invasion and proliferation

and triggers apoptosis. FEBS Letters 566 169–172. (doi:10.1016/j.febslet.

2004.04.025)

Cheng SY, Leonard JL & Davis PJ 2010 Molecular aspects of thyroid hormone

actions. Endocrine Reviews 31 139–170. (doi:10.1210/er.2009-0007)

Conti M & Beavo J 2007 Biochemistry and physiology of cyclic nucleotide

phosphodiesterases: essential components in cyclic nucleotide signaling.

Annual Review of Biochemistry 76 481–511. (doi:10.1146/annurev.biochem.

76.060305.150444)





http://dx.doi.org/10.1016/S0092-8674(00)80691-1

http://dx.doi.org/10.1095/biolreprod.105.043588

http://dx.doi.org/10.1095/biolreprod.105.043588

http://dx.doi.org/10.1016/j.tcb.2007.07.010

http://dx.doi.org/10.1016/S0898-6568(01)00264-9

http://dx.doi.org/10.1074/jbc.M004236200

http://dx.doi.org/10.1124/pr.58.3.5

http://dx.doi.org/10.1124/pr.58.3.5

http://dx.doi.org/10.1530/eje.0.1330451



http://dx.doi.org/10.1007/978-0-387-49310-7_2

http://dx.doi.org/10.1016/j.dnarep.2003.09.007

http://dx.doi.org/10.1093/hmg/ddm019


http://dx.doi.org/10.1093/emboj/19.15.4056

http://dx.doi.org/10.1093/bioinformatics/btp595

http://dx.doi.org/10.1093/bioinformatics/btp595

http://dx.doi.org/10.1586/eem.10.42

http://dx.doi.org/10.1038/sj.emboj.7601636

http://dx.doi.org/10.1074/jbc.272.17.11452



http://dx.doi.org/10.1530/EJE-07-0181

http://dx.doi.org/10.1016/j.tem.2010.02.007

http://dx.doi.org/10.1056/NEJMoa1008020

http://dx.doi.org/10.1016/S1097-2765(05)00020-1



http://dx.doi.org/10.1016/j.febslet.2004.04.025


http://dx.doi.org/10.1210/er.2009-0007

http://dx.doi.org/10.1146/annurev.biochem.76.060305.150444



Cox MB & Miller CA III 2004 Cooperation of heat shock protein 90 and p23

in aryl hydrocarbon receptor signaling. Cell Stress and Chaperones 9 4–20.

(doi:10.1379/460.1)

Daly AF, Vanbellinghen JF, Khoo SK, Jaffrain-Rea ML, Naves LA, Guitelman

MA, Murat A, Emy P, Gimenez-Roqueplo AP, Tamburrano G et al. 2007

Aryl hydrocarbon receptor-interacting protein gene mutations in familial

isolated pituitary adenomas: analysis in 73 families. Journal of Clinical

Endocrinology and Metabolism 92 1891–1896. (doi:10.1210/jc.2006-2513)

Daly AF, Tichomirowa MA, Petrossians P, Heliovaara E, Jaffrain-Rea ML,

Barlier A, Naves LA, Ebeling T, Karhu A, Raappana A et al. 2010 Clinical

characteristics and therapeutic responses in patients with germ-line AIP

mutations and pituitary adenomas: an international collaborative study.

Journal of Clinical Endocrinology and Metabolism 95 E373–E383. (doi:10.

1210/jc.2009-2556)

D’Andrea LD & Regan L 2003 TPR proteins: the versatile helix. Trends in

Biochemical Sciences 28 655–662. (doi:10.1016/j.tibs.2003.10.007)

Das AK, Cohen PW & Barford D 1998 The structure of the tetratricopeptide

repeats of protein phosphatase 5: implications for TPR-mediated protein–

protein interactions. EMBO Journal 17 1192–1199. (doi:10.1093/emboj/

17.5.1192)

Denison MS & Nagy SR 2003 Activation of the aryl hydrocarbon receptor by

structurally diverse exogenous and endogenous chemicals. Annual Review of

Pharmacology and Toxicology 43 309–334. (doi:10.1146/annurev.pharmtox.

43.100901.135828)

Deribe YL, Wild P, Chandrashaker A, Curak J, Schmidt MH, Kalaidzidis Y,

Milutinovic N, Kratchmarova I, Buerkle L, Fetchko MJ et al. 2009

Regulation of epidermal growth factor receptor trafficking by lysine

deacetylase HDAC6. Science Signaling 2 ra84. (doi:10.1126/scisignal.

2000576)

Dietrich C & Kaina B 2010 The aryl hydrocarbon receptor (AhR) in

the regulation of cell–cell contact and tumor growth. Carcinogenesis 31

1319–1328. (doi:10.1093/carcin/bgq028)

Dirnberger D, Messerschmid M & Baumeister R 2008 An optimized split-

ubiquitin cDNA-library screening system to identify novel interactors of

the human Frizzled 1 receptor. Nucleic Acids Research 36 e37. (doi:10.1093/

nar/gkm1163)

Dittmar KD, Demady DR, Stancato LF, Krishna P & Pratt WB 1997 Folding

of the glucocorticoid receptor by the heat shock protein (hsp) 90-based

chaperone machinery. The role of p23 is to stabilize receptor.hsp90

heterocomplexes formed by hsp90.p60.hsp70. Journal of Biological Chemistry

272 21213–21220. (doi:10.1074/jbc.272.34.21213)

Dohi T, Beltrami E, Wall NR, Plescia J & Altieri DC 2004 Mitochondrial

survivin inhibits apoptosis and promotes tumorigenesis. Journal of Clinical

Investigation 114 1117–1127. (doi:10.1172/JCI200422222)

Fields S 2005 High-throughput two-hybrid analysis. The promise and the

peril. FEBS Journal 272 5391–5399. (doi:10.1111/j.1742-4658.2005.

04973.x)

Flaveny C, Perdew GH & Miller CA III 2009 The aryl-hydrocarbon receptor

does not require the p23 co-chaperone for ligand binding and target gene

expression in vivo. Toxicology Letters 189 57–62. (doi:10.1016/j.toxlet.2009.

05.002)

Fortugno P, Beltrami E, Plescia J, Fontana J, Pradhan D, Marchisio PC,

Sessa WC & Altieri DC 2003 Regulation of survivin function by Hsp90.

PNAS 100 13791–13796. (doi:10.1073/pnas.2434345100)

Freeman BC, Felts SJ, Toft DO & Yamamoto KR 2000 The p23 molecular

chaperones act at a late step in intracellular receptor action to differentially

affect ligand efficacies. Genes and Development 14 422–434. (doi:10.1101/

gad.14.4.422)

Froidevaux MS, Berg P, Seugnet I, Decherf S, Becker N, Sachs LM, Bilesimo P,

Nygard M, Pongratz I & Demeneix BA 2006 The co-chaperone XAP2 is

required for activation of hypothalamic thyrotropin-releasing hormone

transcription in vivo. EMBO Reports 7 1035–1039. (doi:10.1038/sj.embor.

7400778)

Fusco D, Vargiolu M, Vidone M, Mariani E, Pennisi LF, Bonora E, Capellari S,

Dirnberger D, Baumeister R, Martinelli P et al. 2010 The RET51/FKBP52

complex and its involvement in Parkinson disease. Human Molecular Genetics

19 2804–2816. (doi:10.1093/hmg/ddq181)


Gadelha MR, Une KN, Rohde K, Vaisman M, Kineman RD & Frohman LA

2000 Isolated familial somatotropinomas: establishment of linkage to

chromosome 11q13.1–11q13.3 and evidence for a potential second locus at

chromosome 2p16–12. Journal of Clinical Endocrinology and Metabolism 85

707–714. (doi:10.1210/jc.85.2.707)

Galat A & Metcalfe SM 1995 Peptidylproline cis/trans isomerases. Progress in

Biophysics and Molecular Biology 63 67–118. (doi:10.1016/0079-

6107(94)00009-X)

Galigniana MD, Harrell JM, Murphy PJ, Chinkers M, Radanyi C, Renoir JM,

Zhang M & Pratt WB 2002 Binding of hsp90-associated immunophilins to

cytoplasmic dynein: direct binding and in vivo evidence that the

peptidylprolyl isomerase domain is a dynein interaction domain.

Biochemistry 41 13602–13610. (doi:10.1021/bi020399z)

Georgitsi M, Raitila A, Karhu A, Tuppurainen K, Makinen MJ, Vierimaa O,

Paschke R, Saeger W, van der Luijt RB, Sane T et al. 2007 Molecular

diagnosis of pituitary adenoma predisposition caused by aryl hydrocarbon

receptor-interacting protein gene mutations. PNAS 104 4101–4105.

(doi:10.1073/pnas.0700004104)

Georgitsi M, De ME, Cannavo S, Makinen MJ, Tuppurainen K, Pauletto P,

Curto L, Weil RJ, Paschke R, Zielinski G et al. 2008 Aryl hydrocarbon

receptor interacting protein (AIP) gene mutation analysis in children

and adolescents with sporadic pituitary adenomas. Clinical Endocrinology 69

621–627. (doi:10.1111/j.1365-2265.2008.03266.x)

Gething MJ & Sambrook J 1992 Protein folding in the cell. Nature 355 33–45.

(doi:10.1038/355033a0)

Goebl M & Yanagida M 1991 The TPR snap helix: a novel protein

repeat motif from mitosis to transcription. Trends in Biochemical Sciences 16

173–177. (doi:10.1016/0968-0004(91)90070-C)

Goldfarb SB, Kashlan OB, Watkins JN, Suaud L, Yan W, Kleyman TR &

Rubenstein RC 2006 Differential effects of Hsc70 and Hsp70 on the

intracellular trafficking and functional expression of epithelial sodium

channels. PNAS 103 5817–5822. (doi:10.1073/pnas.0507903103)

Grad I & Picard D 2007 The glucocorticoid responses are shaped by molecular

chaperones. Molecular and Cellular Endocrinology 275 2–12. (doi:10.1016/

j.mce.2007.05.018)

Haarmann-Stemmann T, Bothe H & Abel J 2009 Growth factors, cytokines

and their receptors as downstream targets of arylhydrocarbon receptor

(AhR) signaling pathways. Biochemical Pharmacology 77 508–520. (doi:10.

1016/j.bcp.2008.09.013)

Hahn ME 2002 Aryl hydrocarbon receptors: diversity and evolution. Chemico-

Biological Interactions 141 131–160. (doi:10.1016/S0009-2797(02)00070-4)

Harari PM, Allen GW & Bonner JA 2007 Biology of interactions:

antiepidermal growth factor receptor agents. Journal of Clinical Oncology 25

4057–4065. (doi:10.1200/JCO.2007.11.8984)

Heitzer MD, Wolf IM, Sanchez ER, Witchel SF & DeFranco DB 2007

Glucocorticoid receptor physiology. Reviews in Endocrine and Metabolic

Disorders 8 321–330. (doi:10.1007/s11154-007-9059-8)

Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J,

Tujague M, Strom A, Treuter E, Warner M et al. 2007 Estrogen receptors:

how do they signal and what are their targets. Physiological Reviews 87

905–931. (doi:10.1152/physrev.00026.2006)

Heliovaara E, Raitila A, Launonen V, Paetau A, Arola J, Lehtonen H, Sane T,

Weil RJ, Vierimaa O, Salmela P et al. 2009 The expression of AIP-related

molecules in elucidation of cellular pathways in pituitary adenomas.

American Journal of Pathology 175 2501–2507. (doi:10.2353/ajpath.2009.

081131)

Heliovaara E, Tuupanen S, Ahlsten M, Hodgson S, De ME, Kuismin O, Izatt L,

McKinlay Gardner RJ, Gundogdu S, Lucassen A et al. 2010 No evidence of

RET germline mutations in familial pituitary adenoma. Journal of Molecular

Endocrinology 46 1–8. (doi:10.1677/JME-10-0052)

Hollingshead BD, Petrulis JR & Perdew GH 2004 The aryl hydrocarbon

(Ah) receptor transcriptional regulator hepatitis B virus X-associated

protein 2 antagonizes p23 binding to Ah receptor–Hsp90 complexes and

is dispensable for receptor function. Journal of Biological Chemistry 279

45652–45661. (doi:10.1074/jbc.M407840200)

Huston E, Beard M, McCallum F, Pyne NJ, Vandenabeele P, Scotland G &

Houslay MD 2000 The cAMP-specific phosphodiesterase PDE4A5 is



http://dx.doi.org/10.1379/460.1

http://dx.doi.org/10.1210/jc.2006-2513

http://dx.doi.org/10.1210/jc.2009-2556

http://dx.doi.org/10.1210/jc.2009-2556

http://dx.doi.org/10.1016/j.tibs.2003.10.007



http://dx.doi.org/10.1146/annurev.pharmtox.43.100901.135828

http://dx.doi.org/10.1146/annurev.pharmtox.43.100901.135828

http://dx.doi.org/10.1126/scisignal.2000576

http://dx.doi.org/10.1126/scisignal.2000576

http://dx.doi.org/10.1093/carcin/bgq028

http://dx.doi.org/10.1093/nar/gkm1163

http://dx.doi.org/10.1093/nar/gkm1163


http://dx.doi.org/10.1172/JCI200422222

http://dx.doi.org/10.1111/j.1742-4658.2005.04973.x

http://dx.doi.org/10.1111/j.1742-4658.2005.04973.x

http://dx.doi.org/10.1016/j.toxlet.2009.05.002

http://dx.doi.org/10.1016/j.toxlet.2009.05.002

http://dx.doi.org/10.1073/pnas.2434345100

http://dx.doi.org/10.1101/gad.14.4.422

http://dx.doi.org/10.1101/gad.14.4.422

http://dx.doi.org/10.1038/sj.embor.7400778

http://dx.doi.org/10.1038/sj.embor.7400778

http://dx.doi.org/10.1093/hmg/ddq181

http://dx.doi.org/10.1210/jc.85.2.707

http://dx.doi.org/10.1016/0079-6107(94)00009-X

http://dx.doi.org/10.1016/0079-6107(94)00009-X

http://dx.doi.org/10.1021/bi020399z


http://dx.doi.org/10.1111/j.1365-2265.2008.03266.x

http://dx.doi.org/10.1038/355033a0

http://dx.doi.org/10.1016/0968-0004(91)90070-C


http://dx.doi.org/10.1016/j.mce.2007.05.018

http://dx.doi.org/10.1016/j.mce.2007.05.018

http://dx.doi.org/10.1016/j.bcp.2008.09.013


http://dx.doi.org/10.1016/S0009-2797(02)00070-4

http://dx.doi.org/10.1200/JCO.2007.11.8984

http://dx.doi.org/10.1007/s11154-007-9059-8

http://dx.doi.org/10.1152/physrev.00026.2006

http://dx.doi.org/10.2353/ajpath.2009.081131


http://dx.doi.org/10.1677/JME-10-0052



cleaved downstream of its SH3 interaction domain by caspase-3.

Consequences for altered intracellular distribution. Journal of Biological

Chemistry 275 28063–28074. (doi:10.1074/jbc.M906144199)

Igreja S, Chahal HS, King P, Bolger GB, Srirangalingam U, Guasti L, Chapple

JP, Trivellin G, Gueorguiev M, Guegan K et al. 2010 Characterization of

aryl hydrocarbon receptor interacting protein (AIP) mutations in familial

isolated pituitary adenoma families. Human Mutation 31 950–960. (doi:10.

1002/humu.21292)

Jaffrain-Rea ML, Angelini M, Gargano D, Tichomirowa MA, Daly AF,

Vanbellinghen JF, D’Innocenzo E, Barlier A, Giangaspero F, Esposito V et al.

2009 Expression of aryl hydrocarbon receptor (AHR) and AHR-

interacting protein in pituitary adenomas: pathological and clinical

implications. Endocrine-Related Cancer 16 1029–1043. (doi:10.1677/ERC-

09-0094)

Jarymowycz VA, Cortajarena AL, Regan L & Stone MJ 2008 Comparison of

the backbone dynamics of a natural and a consensus designed 3-TPR

domain. Journal of Biomolecular NMR 41 169–178. (doi:10.1007/s10858-

008-9250-6)

Jiang LI, Collins J, Davis R, Lin KM, DeCamp D, Roach T, Hsueh R,

Rebres RA, Ross EM, Taussig R et al. 2007 Use of a cAMP BRET

sensor to characterize a novel regulation of cAMP by the sphingosine

1-phosphate/G13 pathway. Journal of Biological Chemistry 282 10576–10584.

(doi:10.1074/jbc.M609695200)

Jiang LI, Collins J, Davis R, Fraser ID & Sternweis PC 2008 Regulation of

cAMP responses by the G12/13 pathway converges on adenylyl cyclase VII.

Journal of Biological Chemistry 283 23429–23439. (doi:10.1074/jbc.

M803281200)

Kang BH & Altieri DC 2006 Regulation of survivin stability by the aryl

hydrocarbon receptor-interacting protein. Journal of Biological Chemistry 281

24721–24727. (doi:10.1074/jbc.M603175200)

Kang BH, Xia F, Pop R, Dohi T, Socolovsky M & Altieri DC 2011

Developmental Control of apoptosis by the immunophilin aryl hydro-

carbon receptor-interacting protein (AIP) involves mitochondrial import of

the survivin protein. The Journal of Biological Chemistry In press. (doi:10.

1074/jbc.M110.210120)

Kashuba E, Kashuba V, Pokrovskaja K, Klein G & Szekely L 2000

Epstein–Barr virus encoded nuclear protein EBNA-3 binds XAP-2,

a protein associated with Hepatitis B virus X antigen.Oncogene 19 1801–1806.

(doi:10.1038/sj.onc.1203501)

Kashuba EV, Gradin K, Isaguliants M, Szekely L, Poellinger L, Klein G &

Kazlauskas A 2006 Regulation of transactivation function of the aryl

hydrocarbon receptor by the Epstein–Barr virus-encoded EBNA-3

protein. Journal of Biological Chemistry 281 1215–1223. (doi:10.1074/jbc.

M509036200)

Kazlauskas A, Poellinger L & Pongratz I 1999 Evidence that the co-chaperone

p23 regulates ligand responsiveness of the dioxin (aryl hydrocarbon)

receptor. Journal of Biological Chemistry 274 13519–13524. (doi:10.1074/jbc.

274.19.13519)

Kazlauskas A, Poellinger L & Pongratz I 2000 The immunophilin-like protein

XAP2 regulates ubiquitination and subcellular localization of the dioxin


M007765200)

Kazlauskas A, Sundstrom S, Poellinger L & Pongratz I 2001 The hsp90

chaperone complex regulates intracellular localization of the dioxin

receptor. Molecular and Cellular Biology 21 2594–2607. (doi:10.1128/MCB.

21.7.2594-2607.2001)

Kazlauskas A, Poellinger L & Pongratz I 2002 Two distinct regions of the

immunophilin-like protein XAP2 regulate dioxin receptor function and

interaction with hsp90. Journal of Biological Chemistry 277 11795–11801.

(doi:10.1074/jbc.M200053200)

Kekatpure VD, Dannenberg AJ & Subbaramaiah K 2009 HDAC6 modulates

Hsp90 chaperone activity and regulates activation of aryl hydrocarbon

receptor signaling. Journal of Biological Chemistry 284 7436–7445. (doi:10.

1074/jbc.M808999200)

Koike K 2009 Hepatitis B virus X gene is implicated in liver carcinogenesis.

Cancer Letters 286 60–68. (doi:10.1016/j.canlet.2009.04.010)


Kuzhandaivelu N, Cong YS, Inouye C, Yang WM & Seto E 1996 XAP2, a

novel hepatitis B virus X-associated protein that inhibits X transactivation.

Nucleic Acids Research 24 4741–4750. (doi:10.1093/nar/24.23.4741)

Laenger A, Lang-Rollin I, Kozany C, Zschocke J, Zimmermann N, Ruegg J,

Holsboer F, Hausch F & Rein T 2009 XAP2 inhibits glucocorticoid

receptor activity in mammalian cells. FEBS Letters 583 1493–1498. (doi:10.

1016/j.febslet.2009.03.072)

Lai AZ, Gujral TS & Mulligan LM 2007 RET signaling in endocrine tumors:

delving deeper into molecular mechanisms. Endocrine Pathology 18 57–67.

(doi:10.1007/s12022-007-0009-5)

Lai ZF, Chen YZ, Feng LP, Meng XM, Ding JF, Wang LY, Ye J, Li P, Cheng

XS, Kitamoto Y et al. 2008 Overexpression of TNNI3K, a cardiac-specific

MAP kinase, promotes P19CL6-derived cardiac myogenesis and prevents

myocardial infarction-induced injury. American Journal of Physiology. Heart

and Circulatory Physiology 295 H708–H716. (doi:10.1152/ajpheart.00252.

2008)

LaPres JJ, Glover E, Dunham EE, Bunger MK & Bradfield CA 2000 ARA9

modifies agonist signaling through an increase in cytosolic aryl hydrocarbon


275.9.6153)

Lennox CA, Trivellin G & Korbonits M 2011 Evaluation of the interaction of

phosphodiesterases 2A and 4A5 with the aryl hydrocarbon receptor

interacting protein in pituitary cells. Endocrine Abstracts 25 P245.

Leontiou CA, Gueorguiev M, van der Spuy J, Quinton R, Lolli F, Hassan S,

Chahal HS, Igreja SC, Jordan S, Rowe J et al. 2008 The role of the aryl

hydrocarbon receptor-interacting protein gene in familial and sporadic

pituitary adenomas. Journal of Clinical Endocrinology and Metabolism 93

2390–2401. (doi:10.1210/jc.2007-2611)

Lin BC, Sullivan R, Lee Y, Moran S, Glover E & Bradfield CA 2007 Deletion

of the aryl hydrocarbon receptor-associated protein 9 leads to cardiac

malformation and embryonic lethality. Journal of Biological Chemistry 282

35924–35932. (doi:10.1074/jbc.M705471200)

Lin DT, Fretier P, Jiang C & Vincent SR 2010 Nitric oxide signaling

via cGMP-stimulated phosphodiesterase in striatal neurons. Synapse 64

460–466. (doi:10.1002/syn.20750)

Lovell SC, Li X, Weerasinghe NR & Hentges KE 2009 Correlation of

microsynteny conservation and disease gene distribution in mammalian

genomes. BMC Genomics 10 521. (doi:10.1186/1471-2164-10-521)

Lugnier C 2006 Cyclic nucleotide phosphodiesterase (PDE) superfamily: a

new target for the development of specific therapeutic agents. Pharmacology

and Therapeutics 109 366–398. (doi:10.1016/j.pharmthera.2005.07.003)

Ma Q & Whitlock JP Jr 1997 A novel cytoplasmic protein that interacts with

the Ah receptor, contains tetratricopeptide repeat motifs, and augments the

transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of

Biological Chemistry 272 8878–8884. (doi:10.1074/jbc.272.14.8878)

Mackenzie KF, Topping EC, Bugaj-Gaweda B, Deng C, Cheung YF, Olsen

AE, Stockard CR, High ML, Baillie GS, Grizzle WE et al. 2008 Human

PDE4A8, a novel brain-expressed PDE4 cAMP-specific phosphodiesterase

that has undergone rapid evolutionary change. Biochemical Journal 411

361–369. (doi:10.1042/BJ20071251)

Meyer BK & Perdew GH 1999 Characterization of the AhR–hsp90–XAP2

core complex and the role of the immunophilin-related protein XAP2 in

AhR stabilization. Biochemistry 38 8907–8917. (doi:10.1021/bi982223w)

Meyer BK, Pray-Grant MG, Vanden Heuvel JP & Perdew GH 1998 Hepatitis

B virus X-associated protein 2 is a subunit of the unliganded aryl

hydrocarbon receptor core complex and exhibits transcriptional enhancer

activity. Molecular and Cellular Biology 18 978–988.

Meyer BK, Petrulis JR & Perdew GH 2000 Aryl hydrocarbon (Ah) receptor

levels are selectively modulated by hsp90-associated immunophilin

homolog XAP2. Cell Stress & Chaperones 5 243–254. (doi:10.1379/1466-

1268(2000)005!0243:AHARLAO2.0.CO;2)

Montanana CF, Daly AF, Tichomirowa MA, Vanbellinghen JF, Jaffrain-Rea

ML, Trescoli Serrano C, Riesgo Suares P, Gomez Vela J, Tenes S, Bours V

et al. 2009 TSH-secreting pituitary adenoma in a male patient with a novel

missense AIP mutation. Proceedings of the 91st Annual Meeting of the Endocrine

Society P1668.

Morales JL & Perdew GH 2007 Carboxyl terminus of hsc70-interacting

protein (CHIP) can remodel mature aryl hydrocarbon receptor (AhR)




http://dx.doi.org/10.1002/humu.21292

http://dx.doi.org/10.1002/humu.21292

http://dx.doi.org/10.1677/ERC-09-0094

http://dx.doi.org/10.1677/ERC-09-0094

http://dx.doi.org/10.1007/s10858-008-9250-6

http://dx.doi.org/10.1007/s10858-008-9250-6





http://dx.doi.org/10.1074/jbc.M110.210120


http://dx.doi.org/10.1038/sj.onc.1203501







http://dx.doi.org/10.1128/MCB.21.7.2594-2607.2001

http://dx.doi.org/10.1128/MCB.21.7.2594-2607.2001




http://dx.doi.org/10.1016/j.canlet.2009.04.010

http://dx.doi.org/10.1093/nar/24.23.4741



http://dx.doi.org/10.1007/s12022-007-0009-5

http://dx.doi.org/10.1152/ajpheart.00252.2008

http://dx.doi.org/10.1152/ajpheart.00252.2008



http://dx.doi.org/10.1210/jc.2007-2611


http://dx.doi.org/10.1002/syn.20750

http://dx.doi.org/10.1186/1471-2164-10-521

http://dx.doi.org/10.1016/j.pharmthera.2005.07.003


http://dx.doi.org/10.1042/BJ20071251

http://dx.doi.org/10.1021/bi982223w

http://dx.doi.org/10.1379/1466-1268(2000)005<0243:AHARLA>2.0.CO;2

http://dx.doi.org/10.1379/1466-1268(2000)005<0243:AHARLA>2.0.CO;2


complexes and mediate ubiquitination of both the AhR and the 90 kDa

heat-shock protein (hsp90) in vitro. Biochemistry 46 610–621. (doi:10.1021/

bi062165b)

Nair SC, Toran EJ, Rimerman RA, Hjermstad S, Smithgall TE & Smith DF

1996 A pathway of multi-chaperone interactions common to diverse

regulatory proteins: estrogen receptor, Fes tyrosine kinase, heat shock

transcription factor Hsf1, and the aryl hydrocarbon receptor. Cell Stress

and Chaperones 1 237–250. (doi:10.1379/1466-1268(1996)001!0237:APOMCIO2.3.CO;2)

Nakata A, Urano D, Fujii-Kuriyama Y, Mizuno N, Tago K & Itoh H 2009

G-protein signalling negatively regulates the stability of aryl hydrocarbon

receptor. EMBO Reports 10 622–628. (doi:10.1038/embor.2009.35)

Nguyen LP & Bradfield CA 2008 The search for endogenous activators of the

aryl hydrocarbon receptor. Chemical Research in Toxicology 21 102–116.

(doi:10.1021/tx7001965)

Nilsson S & Gustafsson JA 2011 Estrogen receptors: therapies targeted to

receptor subtypes. Clinical Pharmacology and Therapeutics 89 44–55. (doi:10.

1038/clpt.2010.226)

Nukaya M, Lin BC, Glover E, Moran SM, Kennedy GD & Bradfield CA

2010 The aryl hydrocarbon receptor-interacting protein (AIP) is required

for dioxin-induced hepatotoxicity but not for the induction of the Cyp1a1

and Cyp1a2 genes. Journal of Biological Chemistry 285 35599–35605. (doi:10.

1074/jbc.M110.132043)

Nyarko A, Mosbahi K, Rowe AJ, Leech A, Boter M, Shirasu K & Kleanthous

C 2007 TPR-mediated self-association of plant SGT1. Biochemistry 46

11331–11341. (doi:10.1021/bi700735t)

O’Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W,

Bolger G, Frame M & Houslay MD 1996 The SH3 domain of Src tyrosyl

protein kinase interacts with the N-terminal splice region of the PDE4A

cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochemical

Journal 318 255–261.

Oesch-Bartlomowicz B & Oesch F 2009 Role of cAMP in mediating AHR

signaling. Biochemical Pharmacology 77 627–641. (doi:10.1016/j.bcp.2008.

10.017)

Oesch-Bartlomowicz B, Huelster A, Wiss O, Antoniou-Lipfert P, Dietrich C,

Arand M, Weiss C, Bockamp E & Oesch F 2005 Aryl hydrocarbon receptor

activation by cAMP vs. dioxin: divergent signaling pathways. PNAS 102

9218–9223. (doi:10.1073/pnas.0503488102)

Ohtake F, Baba A, Takada I, Okada M, Iwasaki K, Miki H, Takahashi S,

Kouzmenko A, Nohara K, Chiba T et al. 2007 Dioxin receptor is a ligand-

dependent E3 ubiquitin ligase. Nature 446 562–566. (doi:10.1038/

nature05683)

Ohtake F, Fujii-Kuriyama Y & Kato S 2009 AhR acts as an E3 ubiquitin

ligase to modulate steroid receptor functions. Biochemical Pharmacology 77

474–484. (doi:10.1016/j.bcp.2008.08.034)

de Oliveira SK & Smolenski A 2009 Phosphodiesterases link the aryl

hydrocarbon receptor complex to cyclic nucleotide signaling. Biochemical

Pharmacology 77 723–733. (doi:10.1016/j.bcp.2008.08.027)

de Oliveira SK, Hoffmeister M, Gambaryan S, Muller-Esterl W, Guimaraes JA

& Smolenski AP 2007 Phosphodiesterase 2A forms a complex with the

co-chaperone XAP2 and regulates nuclear translocation of the aryl

hydrocarbon receptor. Journal of Biological Chemistry 282 13656–13663.

(doi:10.1074/jbc.M610942200)

Owens-Grillo JK, Stancato LF, Hoffmann K, Pratt WB & Krishna P 1996

Binding of immunophilins to the 90 kDa heat shock protein (hsp90) via a

tetratricopeptide repeat domain is a conserved protein interaction in plants.

Biochemistry 35 15249–15255. (doi:10.1021/bi9615349)

Pearl LH & Prodromou C 2006 Structure and mechanism of the Hsp90

molecular chaperone machinery. Annual Review of Biochemistry 75 271–294.

(doi:10.1146/annurev.biochem.75.103004.142738)

Perdew GH 1988 Association of the Ah receptor with the 90-kDa heat shock

protein. Journal of Biological Chemistry 263 13802–13805.

Petrulis JR, Hord NG & Perdew GH 2000 Subcellular localization of the aryl

hydrocarbon receptor is modulated by the immunophilin homolog

hepatitis B virus X-associated protein 2. Journal of Biological Chemistry 275

37448–37453. (doi:10.1074/jbc.M006873200)

Petrulis JR, Kusnadi A, Ramadoss P, Hollingshead B & Perdew GH 2003 The

hsp90 Co-chaperone XAP2 alters importin beta recognition of the bipartite


nuclear localization signal of the Ah receptor and represses transcriptional

activity. Journal of Biological Chemistry 278 2677–2685. (doi:10.1074/jbc.

M209331200)

Pollenz RS & Dougherty EJ 2005 Redefining the role of the endogenous

XAP2 and C-terminal hsp70-interacting protein on the endogenous Ah

receptors expressed in mouse and rat cell lines. Journal of Biological Chemistry

280 33346–33356. (doi:10.1074/jbc.M506619200)

Pollenz RS, Wilson SE & Dougherty EJ 2006 Role of endogenous XAP2

protein on the localization and nucleocytoplasmic shuttling of the

endogenous mouse Ahb-1 receptor in the presence and absence of ligand.

Molecular Pharmacology 70 1369–1379. (doi:10.1124/mol.106.027672)

Pongratz I, Ruegg J, Swedenborg E & Kramarova T 2009 Molecular

mechanisms of AhR and NR crosstalk. Endocrine Abstracts 20 S24.2.

Pratt WB, Galigniana MD, Harrell JM & DeFranco DB 2004 Role of hsp90

and the hsp90-binding immunophilins in signalling protein movement.

Cellular Signalling 16 857–872. (doi:10.1016/j.cellsig.2004.02.004)

Raitila A, Lehtonen HJ, Arola J, Heliovaara E, Ahlsten M, Georgitsi M,

Jalanko A, Paetau A, Aaltonen LA & Karhu A 2010 Mice with inactivation

of aryl hydrocarbon receptor-interacting protein (Aip) display complete

penetrance of pituitary adenomas with aberrant ARNT expression.

American Journal of Pathology 177 1969–1976. (doi:10.2353/ajpath.2010.

100138)

Ramadoss P, Petrulis JR, Hollingshead BD, Kusnadi A & Perdew GH 2004

Divergent roles of hepatitis B virus X-associated protein 2 (XAP2) in

human versus mouse Ah receptor complexes. Biochemistry 43 700–709.

(doi:10.1021/bi035827v)

Rosman GJ, Martins TJ, Sonnenburg WK, Beavo JA, Ferguson K &

Loughney K 1997 Isolation and characterization of human cDNAs

encoding a cGMP-stimulated 3 0,5 0-cyclic nucleotide phosphodiesterase.

Gene 191 89–95. (doi:10.1016/S0378-1119(97)00046-2)

Russell LC, Whitt SR, Chen MS & Chinkers M 1999 Identification of

conserved residues required for the binding of a tetratricopeptide repeat

domain to heat shock protein 90. Journal of Biological Chemistry 274

20060–20063. (doi:10.1074/jbc.274.29.20060)

Sadhu K, Hensley K, Florio VA & Wolda SL 1999 Differential expression of

the cyclic GMP-stimulated phosphodiesterase PDE2A in human venous

and capillary endothelial cells. Journal of Histochemistry and Cytochemistry 47

895–906. (doi:10.1177/002215549904700707)

Scheufler C, Brinker A, Bourenkov G, Pegoraro S, Moroder L, Bartunik H,

Hartl FU & Moarefi I 2000 Structure of TPR domain–peptide complexes:

critical elements in the assembly of the Hsp70–Hsp90 multichaperone

machine. Cell 101 199–210. (doi:10.1016/S0092-8674(00)80830-2)

Schulke JP, Wochnik GM, Lang-Rollin I, Gassen NC, Knapp RT, Berning B,

Yassouridis A & Rein T 2010 Differential impact of tetratricopeptide repeat

proteins on the steroid hormone receptors. PLoS ONE 5 e11717. (doi:10.

1371/journal.pone.0011717)

Silverstein AM, Galigniana MD, Chen MS, Owens-Grillo JK, Chinkers M &

Pratt WB 1997 Protein phosphatase 5 is a major component of

glucocorticoid receptor.hsp90 complexes with properties of an FK506-

binding immunophilin. Journal of Biological Chemistry 272 16224–16230.

(doi:10.1074/jbc.272.26.16224)

Smith DF 2004 Tetratricopeptide repeat cochaperones in steroid receptor

complexes. Cell Stress and Chaperones 9 109–121. (doi:10.1379/CSC-31.1)

Smith DF, Whitesell L, Nair SC, Chen S, Prapapanich V & Rimerman RA

1995 Progesterone receptor structure and function altered by geldanamy-

cin, an hsp90-binding agent. Molecular and Cellular Biology 15 6804–6812.

Snider J, Kittanakom S, Curak J & Stagljar I 2010 Split-ubiquitin based

membrane yeast two-hybrid (MYTH) system: a powerful tool for

identifying protein–protein interactions. Journal of Visualized Experiments

36 1698. (doi:10.3791/1698)

Soares BS, Eguchi K & Frohman LA 2005 Tumor deletion mapping on

chromosome 11q13 in eight families with isolated familial somatotropi-

noma and in 15 sporadic somatotropinomas. Journal of Clinical Endocrinology

and Metabolism 90 6580–6587. (doi:10.1210/jc.2005-1478)

Stephenson DT, Coskran TM, Wilhelms MB, Adamowicz WO, O’Donnell

MM, Muravnick KB, Menniti FS, Kleiman RJ & Morton D 2009



http://dx.doi.org/10.1021/bi062165b

http://dx.doi.org/10.1021/bi062165b

http://dx.doi.org/10.1379/1466-1268(1996)001%3C0237:APOMCI%3E2.3.CO;2

http://dx.doi.org/10.1379/1466-1268(1996)001%3C0237:APOMCI%3E2.3.CO;2

http://dx.doi.org/10.1038/embor.2009.35

http://dx.doi.org/10.1021/tx7001965

http://dx.doi.org/10.1038/clpt.2010.226

http://dx.doi.org/10.1038/clpt.2010.226



http://dx.doi.org/10.1021/bi700735t




http://dx.doi.org/10.1038/nature05683

http://dx.doi.org/10.1038/nature05683




http://dx.doi.org/10.1021/bi9615349






http://dx.doi.org/10.1124/mol.106.027672

http://dx.doi.org/10.1016/j.cellsig.2004.02.004



http://dx.doi.org/10.1021/bi035827v

http://dx.doi.org/10.1016/S0378-1119(97)00046-2


http://dx.doi.org/10.1177/002215549904700707

http://dx.doi.org/10.1016/S0092-8674(00)80830-2

http://dx.doi.org/10.1371/journal.pone.0011717

http://dx.doi.org/10.1371/journal.pone.0011717


http://dx.doi.org/10.1379/CSC-31.1

http://dx.doi.org/10.3791/1698

http://dx.doi.org/10.1210/jc.2005-1478


Immunohistochemical localization of phosphodiesterase 2A in multiple

mammalian species. Journal of Histochemistry and Cytochemistry 57 933–949.

(doi:10.1369/jhc.2009.953471)

Stork PJ & Schmitt JM 2002 Crosstalk between cAMP and MAP kinase

signaling in the regulation of cell proliferation. Trends in Cell Biology 12

258–266. (doi:10.1016/S0962-8924(02)02294-8)

Stratakis CA, Tichomirowa MA, Boikos S, Azevedo MF, Lodish M, Martari

M, Verma S, Daly AF, Raygada M, Keil MF et al. 2010 The role of germline

AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing

pituitary adenomas in a large cohort of children, adolescents, and patients

with genetic syndromes. Clinical Genetics 78 457–463. (doi:10.1111/j.1399-

0004.2010.01406.x)

Sumanasekera WK, Tien ES, Turpey R, Vanden Heuvel JP & Perdew GH

2003 Evidence that peroxisome proliferator-activated receptor alpha is

complexed with the 90-kDa heat shock protein and the hepatitis virus B

X-associated protein 2. Journal of Biological Chemistry 278 4467–4473.

(doi:10.1074/jbc.M211261200)

Tahira T, Ishizaka Y, Itoh F, Sugimura T & Nagao M 1990 Characterization of

ret proto-oncogene mRNAs encoding two isoforms of the protein product

in a human neuroblastoma cell line. Oncogene 5 97–102.

Taylor P, Dornan J, Carrello A, Minchin RF, Ratajczak T & Walkinshaw MD

2001 Two structures of cyclophilin 40: folding and fidelity in the TPR

domains. Structure 9 431–438. (doi:10.1016/S0969-2126(01)00603-7)

Tomkinson B, Robertson E & Kieff E 1993 Epstein–Barr virus nuclear

proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth

transformation. Journal of Virology 67 2014–2025.

Vargiolu M, Fusco D, Kurelac I, Dirnberger D, Baumeister R, Morra I,

Melcarne A, Rimondini R, Romeo G & Bonora E 2009 The tyrosine

kinase receptor RET interacts in vivo with aryl hydrocarbon receptor-

interacting protein to alter survivin availability. Journal of Clinical

Endocrinology and Metabolism 94 2571–2578. (doi:10.1210/jc.2008-1980)

Velardez MO, De LA, del Carmen DM, Lasaga M, Pisera D, Seilicovich A &

Duvilanski BH 2000 Role of phosphodiesterase and protein kinase G on

nitric oxide-induced inhibition of prolactin release from the rat anterior

pituitary. European Journal of Endocrinology 143 279–284. (doi:10.1530/eje.0.

1430279)

Verdecia MA, Huang H, Dutil E, Kaiser DA, Hunter T & Noel JP 2000

Structure of the human anti-apoptotic protein survivin reveals a dimeric

arrangement. Nature Structural Biology 7 602–608. (doi:10.1038/77929)

Vierimaa O, Georgitsi M, Lehtonen R, Vahteristo P, Kokko A, Raitila A,

Tuppurainen K, Ebeling TM, Salmela PI, Paschke R et al. 2006 Pituitary

adenoma predisposition caused by germline mutations in the AIP gene.

Science 312 1228–1230. (doi:10.1126/science.1126100)

Vlotides G, Siegel E, Donangelo I, Gutman S, Ren SG & Melmed S 2008

Rat prolactinoma cell growth regulation by epidermal growth factor

receptor ligands. Cancer Research 68 6377–6386. (doi:10.1158/0008-5472.

CAN-08-0508)


Voegel JJ, Heine MJ, Tini M, Vivat V, Chambon P & Gronemeyer H 1998

The coactivator TIF2 contains three nuclear receptor-binding motifs and

mediates transactivation through CBP binding-dependent and -indepen-

dent pathways. EMBO Journal 17 507–519. (doi:10.1093/emboj/17.2.507)

Wei Y, Fan T & Yu M 2008 Inhibitor of apoptosis proteins and apoptosis. Acta

Biochimica et Biophysica Sinica 40 278–288. (doi:10.1111/j.1745-7270.2008.

00407.x)

Worzfeld T, Wettschureck N & Offermanns S 2008 G(12)/G(13)-mediated

signalling in mammalian physiology and disease. Trends in Pharmacological

Sciences 29 582–589. (doi:10.1016/j.tips.2008.08.002)

Yamaguchi Y, Katoh H, Mori K & Negishi M 2002 Galpha(12) and

Galpha(13) interact with Ser/Thr protein phosphatase type 5 and stimulate

its phosphatase activity. Current Biology 12 1353–1358. (doi:10.1016/

S0960-9822(02)01034-5)

Yang D, Welm A & Bishop JM 2004 Cell division and cell survival in the

absence of survivin. PNAS 101 15100–15105. (doi:10.1073/pnas.

0406665101)

Yang J, Roe SM, Cliff MJ, Williams MA, Ladbury JE, Cohen PT & Barford D

2005 Molecular basis for TPR domain-mediated regulation of protein

phosphatase 5. EMBO Journal 24 1–10. (doi:10.1038/sj.emboj.7600496)

Yano M, Terada K & Mori M 2003 AIP is a mitochondrial import mediator

that binds to both import receptor Tom20 and preproteins. Journal of Cell

Biology 163 45–56. (doi:10.1083/jcb.200305051)

Yoon M 2009 The role of PPARalpha in lipid metabolism and obesity:

focusing on the effects of estrogen on PPARalpha actions. Pharmacological

Research 60 151–159. (doi:10.1016/j.phrs.2009.02.004)

Young JC, Hoogenraad NJ & Hartl FU 2003 Molecular chaperones Hsp90

and Hsp70 deliver preproteins to the mitochondrial import receptor

Tom70. Cell 112 41–50. (doi:10.1016/S0092-8674(02)01250-3)

Zaccolo M & Movsesian MA 2007 cAMP and cGMP signaling cross-talk: role

of phosphodiesterases and implications for cardiac pathophysiology.

Circulation Research 100 1569–1578. (doi:10.1161/CIRCRESAHA.106.

144501)

Zeng Q, Chen G, Vlantis A, Tse G & van HC 2008 The contributions of

oestrogen receptor isoforms to the development of papillary and anaplastic

thyroid carcinomas. Journal of Pathology 214 425–433. (doi:10.1002/path.

2297)

Zhao Y, Meng XM, Wei YJ, Zhao XW, Liu DQ, Cao HQ, Liew CC &

Ding JF 2003 Cloning and characterization of a novel cardiac-specific

kinase that interacts specifically with cardiac troponin I. Journal of

Molecular Medicine 81 297–304. (doi:10.1007/s00109-003-0427-x)

Received in final form 21 March 2011Accepted 21 March 2011Made available online as an Accepted Preprint31 March 2011



http://dx.doi.org/10.1369/jhc.2009.953471

http://dx.doi.org/10.1016/S0962-8924(02)02294-8

http://dx.doi.org/10.1111/j.1399-0004.2010.01406.x

http://dx.doi.org/10.1111/j.1399-0004.2010.01406.x


http://dx.doi.org/10.1016/S0969-2126(01)00603-7

http://dx.doi.org/10.1210/jc.2008-1980



http://dx.doi.org/10.1038/77929

http://dx.doi.org/10.1126/science.1126100

http://dx.doi.org/10.1158/0008-5472.CAN-08-0508

http://dx.doi.org/10.1158/0008-5472.CAN-08-0508


http://dx.doi.org/10.1111/j.1745-7270.2008.00407.x

http://dx.doi.org/10.1111/j.1745-7270.2008.00407.x

http://dx.doi.org/10.1016/j.tips.2008.08.002

http://dx.doi.org/10.1016/S0960-9822(02)01034-5

http://dx.doi.org/10.1016/S0960-9822(02)01034-5



http://dx.doi.org/10.1038/sj.emboj.7600496

http://dx.doi.org/10.1083/jcb.200305051

http://dx.doi.org/10.1016/j.phrs.2009.02.004

http://dx.doi.org/10.1016/S0092-8674(02)01250-3

http://dx.doi.org/10.1161/CIRCRESAHA.106.144501

http://dx.doi.org/10.1161/CIRCRESAHA.106.144501

http://dx.doi.org/10.1002/path.2297

http://dx.doi.org/10.1002/path.2297

http://dx.doi.org/10.1007/s00109-003-0427-x

Documents

Practice Guidelines for the Perioperative Management of