Proteins Accompanying the Estrogen Receptor α and β: A Model for Studying Protein Hetero-Complexes

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  • Biocutulysis and Biotrunsfomation, Vol. 19, pp. 4271142 Reprints available directly from the publisher Photocopying permitted by license only

    0 2001 OPA (Overseas Publishers Association) N.V. Published by license under

    the Hanvocd Academic Publishers imprint, part of Gordon and Breach Publishing

    a member of the Taylor & Francis Group. All rights reserved.

    PROTEINS ACCOMPANYING THE ESTROGEN RECEPTOR a AND p: A MODEL FOR STUDYING

    PROTEIN HETERO-COMPLEXES

    ELISABETH JISAa, KLAUS GRAUMANNb and L O I S JUNGBAUERa*

    aht i tUte of Applied Microbiology, University of Agricultural Sciences, Muthgasse 18, Vienna A-1190, Austria; bBiochemie GmbH, Kundl, Austria

    (Received 6 April 2000; Revised 15 November 2000)

    Estrogen receptor a forms a highly dynamic protein complex in its activated and inactivated state. The protein is complexed with heat shock proteins and other accompanying proteins. Upon ligand binding these proteins are shed off, the receptor dimerizes and forms a preinitiation complex with coactivators andlor corepressors. A plethora of proteins has been discovered, associated with this complex. These different proteins may either up- or down-regulate estrogen receptor-mediated transcription of target genes. Real time-biosensor technology is one approach to assessing these extremely dynamic protein complex formations.

    Keywords: Estrogen; Immunophilins; Ligand Hetero-complexes

    INTRODUCTION

    Estrogen receptors belong to the family of steroid hormone receptors(SHR) (Evans, 1988). They play extremely important roles in growth and development, reproduction and hormone dependent cancers such as breast cancer. The receptors are associated with other proteins in their active, ligand-friendly state. Upon activation at least some of these proteins are shed off, the receptor dimerizes and is, if not already located there, transported into the nucleus to form an active transcription complex with other proteins. Functionality and fate of the receptor is highly determined by protein hetero-complexes (Graumann and

    *Corresponding author. Fax: +43-136006-1249. E-mail: jungbaue@hpOl .boku.ac.at

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  • 428 E. JISA et 01.

    Jungbauer. 2000). Since the receptor itself is an extremely labile molecule it is very tedious to produce sufficient quantities for biochemical studies. The development of modem real-time biosensor techniques enabled the access to preliminary data on stability and thermodynamic properties of these hetero- complexes. In this review we describe the nature of the hetero-complex of unligandedinactive as well as ligandedactivated receptor and the methodology to measure these complexes using real-time biosensor technology.

    Like any other members of the nuclear receptor superfamily estrogen receptor a and P (ERa and ERP) are composed of five different domains, which may differ in amino acid homology, but have similar function. The N-terminal A B domain is highly variable in sequence and length. It contains a ligand- independent transactivation function (AF- 1) which activates target gene transcription by directly interacting with components of the transcription machinery or with coactivators that mediate signaling to downstream proteins (Tora et al., 1989; Webb et al., 1998; Tremblay et al., 1999). The C domain comprises DNA binding function and dimerization sequences. Two zinc fingers are responsible for specific interaction between receptor molecules and estrogen response elements. The E domain is responsible for ligand binding, nuclear localization and ligand-dependent transactivation (AF-2) (Webster et al., 1988). The hinge domain between domains C and E contributes flexibility, and represents an anchor for corepressor proteins. The C-terminal F domain also contributes to transactivation capacity.

    In 1996 a novel form of the estrogen receptor was discovered (Kuiper et al., 1996; Mosselman et al., 1996; Ogawa et al., 1998) and is referred to as ERP with the original form now known as ERa. ERP is able to partially substitute for the function of ERa in ERa knock out mice (Krege et al., 1998; Couse et al., 1999).

    PROTEINS COMPLEXED WITH UNLIGANDED ERa AND ERP

    As with many other intracellular proteins, SHR, such as ERa and ERP, form multi-protein complexes with members of the heat shock and immunophilin protein families (Pratt et al., 1996; Fratt and Toft, 1997; Pratt, 1998). In their ligand free state, SHRs are found associated with hsp90 and the immunophilins FKBP52 or cyclophilin 40 (Cyp-40), as well as other proteins. Although it should be mentioned that not all of the proteins listed below have been detected in unliganded ERa or ERP hetero-complexes, most concepts seem to be true-at least for cytosolic ER.

    Not surprisingly, one major role of these proteins is related to protein folding during receptor synthesis (Rutherford and Zuker, 1994). A core chaperone

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  • PROTEINS ACCOMPANYING THE ESTROGEN RECEPTOR a AND J3 429

    complex formed of hsp90, p60 (Hop-heat shock protein organizing protein) and hsp70 binds nascent polypeptides (Chen et al., 1996). Other proteins like p23, hsp40 or p48 (Hip-heat shock interacting protein) are also involved (Johnson and Toft, 1995; Prapapanich et al., 1996; Kosano et al., 1998). In general, these protein hetero-complexes are highly dynamic, members are partly interchange- able. p60 or hsp70 have been detected in early stages of receptor complex formation while immunophilins replace them during a process called maturation. During this maturation process receptor molecules are not simply folded but transformed into a ligand-friendly state (Freeman et al., 1996).

    Some aspects of receptor translocation from the cytosol to the nucleus and possible recycling are still rather unclear. Despite the high level of complexity due to the number of players, some light has been shed on hetero-complex formatiodcomposition and possible roles of individual complex members during the last years. Table I lists members of receptor hetero-complexes and their possible functions.

    Hsp90 is one of the most abundantly expressed proteins in the cell and plays important roles in protein folding, translocation and also in cell signaling (Pratt, 1998). It forms the core of all known receptor hetero-complexes and is a pre- requisite for high affinity ligand binding. The stoichiometric ratio of hsp90 nuclear receptor molecule interaction appears to be two hsp90 molecules to each nuclear receptor molecule (Segnitz and Gehring, 1995). It is generally accepted that hsp90 is a nucleotide binding protein (Grenert et al., 1999). Graumann and Jungbauer (2000) have recently demonstrated that ATP changes the cooperativity of interaction between hsp90 and other relevant proteins, such as Hop or FKBP51 and FKBP52.

    Hop is a member of early receptor hetero-complexes and, since hsp90 and hsp70 do not directly interact with each other, it forms a molecular bridge between these two chaperones. However, very recently Hop has been found to be

    TABLE I Overview of biological functions of proteins accompanying unliganded SHRs

    Protein Nucleotide Immuno suppression Cell-type Role in Role in Protein folding binding drug binding specijc action translocation transactiwation

    hsp90 yes yes Yes ?

    P60 FKBP51FKBP52 (yes) Yes Yes Yes cyp-40 Yes Yes ? hsp40 yes (?I P23 1 ? ?

    hsp70 yes yes

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  • 330 E. JISA et nl.

    not essential for the conversion of steroid receptors into hetero-complexes and a ligand binding state (Morishima et al., 2000).

    Hsp70 itself has been found in early receptor complexes. Nucleotide binding modulates the interaction with substrate polypeptides. So far, its role in receptor hetero-complexes seems to be related to protein folding. However, as for hsp90, other functional proteins interact with the hetero-complex via hsp70. In this context, cofactors like BAG- 1 have been identified as modulators for chaperone activity (Kanelakis er al., 1999).

    Immunophilins (FKBP.5 1, Fl(BP52, Cyp-40) possess peptidyl-prolyl isomer- ase function and therefore protein folding activity. They have also been linked to receptor shuttling in the cell (Pratt et al.. 1999). Another highly interesting feature of this class of proteins is that they are targets for immunosuppressant drugs such as rapamycin, FK506 or cyclosporin A. Therefore, these proteins form a crosslink to immunological function. Immunophilins, p60 (Hop) and hsp90 molecules interact via tetratricopeptide repeat (TPR) motifs with each other (Carrello et al., 1999). These TPR motifs are formed by a different number of rather distant amino acid patches.

    More recently, p23 has been identified as a member of receptor hetero- complexes. Additionally this hsp90-binding protein has been linked to the ligand activation process (Kazlauskas el al., 1999) and DNA-receptor complex formation and therefore with transcription (Freeman et al., 2000). Hsp40 has been debated as an essential protein for receptor hetero-complex formation. A recent study with purified hsp90 and hsp70 showed that, as for Hop, hsp40 is not essential for hetero-complex formation. The multitude of proteins with different and rather diverse properties makes it clear that investigation of receptor hetero- complexes is a tool to learn more about functional cellular networks.

    COFACTORS OF ER (Y AND f3

    After activation by a hormone or hormone mimic the hetero-complex dissociates. Estrogen receptors stimulate gene transcription assembling into a stable preinitiation complex associated with basal transcription factors like TATA- binding protein (TBP) and TFIIB in order to serve as bridging proteins to RNA polymerase I1 (Horwitz er al., 1996; Smith et al., 1996 and references therein). For efficient hormone-controlled transcription a further class of proteins is required-the so-called coactivators and corepressors. Depending upon the cell type various coactivators and corepressors are present, the final composition of preinitiation complexes will depend on the composition of cofactors present in a given cell. A common characteristic for nuclear coactivator proteins is the

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  • PROTEINS ACCOMPANYING THE ESTROGEN RECEPTOR a AND p 43 1

    signature sequence LXXLL mediating efficient and sufficient binding to liganded nuclear receptors (Heery et al., 1997). Prominent representatives of coactivators belong to the SRC-family consisting of SRC-1, TIF-2/GRIPl and RAC3/ACTR/pCIP/AIB- 1 (Leo and Chen, 2000).

    Steroid receptor coactivator-1 (SRC-1) was shown to enhance transcription by ERa bound to ligands with mixed agonisthtagonist properties, but to inactivate ERa occupied by pure antiestrogens. Members of the SRC-family recruit additional coactivators such as p300/CBP in order to enhance transactivation (Onate et al., 1995; Horwitz et al., 1996; Leo and Chen, 2000). SRC-1 is a genuine coactivator for steroid receptor target gene expression acting by direct contact with receptor molecules (Onate et al., 1995). In contrast SRC-1 enhances transactivation by ERP both in the presence and absence of hormone suggesting distinct molecular mechanisms regulating the transcriptional activity of the two receptor subtypes. In vitro phosphorylation of AF-1 by MAP kinases leads to the recruitment of SRC-1 by ERP (Tremblay et al., 1997; Tremblay et al., 1999).

    Another two proteins mediating the functions of activation function AF-2 are TIF-la and TIF-2. Like other coactivators TIF-la is able to enhance ER- dependent transcription in the presence of hormone by interacting with activation function AF-2. This interaction is selective for certain members of the nuclear receptor family including ER, TR, RAR, RXR and VDR (ThCnot et al., 1997). In the absence of hormone TIF-la interacts better with ERP independently of the presence of estrogen response element (ThCnot et al., 1997). TIF-2 could be characterized to act similar to TIF-1 exhibiting typical characteristics of nuclear receptor coactivators (Voegel et al., 1996).

    A transcriptional coactivator for the C-terminal transactivation domain is glucocorticoid receptor interacting protein 1 (GRIPl), presumed to be the ortholog to human TIF-2. It binds to the ligand binding domain of steroid receptors in the presence but not in the absence of hormone, acting as a transcriptional coactivator for steroid receptors (Hong et al., 1997; Norris et al., 1998).

    Receptor-associated coactivator 3 (RAC3) is highly related to SRC-1 and TIF- 2, enhancing ligand-dependent transcriptional activation. In addition it contains a transcriptional activation domain. (Li et al., 1997). RAC3 was found to activate transcription of several nuclear receptors, including the recently cloned estrogen receptor beta, exhibiting different alpha-helical LXXLL motifs of RAC3 for interaction (Leo et al., 2000).

    Another member of the SRC-1 family is represented by the coactivator amplified in breast cancer-1 (AIB-1). AIB- 1 over-expression and amplification could be observed in a majority of ERa-positive breast and ovarian cancer cell lines. AIB-1 is able to interact with ERa in a ligand-dependent manner and to

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  • 432 E. JISA et ul.

    enhance target gene transcription (Anzick et al., 1997). AIB-1 is suspected of inducing estrogen-independent proliferation of breast and ovarian tumors when present in cell lines expressing mutated ERa binding AIB-1 or other members of the SRC-family hormone-independently. AIB-1 is able to interact with the ligand binding domain of a constitutively active ERa in vitro and to activate basal transcription when cotransfected (Eng et al., 1998).

    In 1995 yet another coactivator RIP140 was characterized to interact with the AF-2 domain of ERor in virro. This interaction can be enhanced in the presence of estrogens but not of antiestrogens in vitro and in vivo (Cavailles et al., 1994; Cavailles et al., 1995). RIP140 was shown to block the stimulatory effect of TIF-2 on ER function suggesting a competitive mode of action of various coactivators. Further RIP140 may act as a negative regulatory factor in target gene transactivation. A remarkable observation was the fact that RIP140 can interact with the ligand binding domain of ERa in the presence of the pure antiestrogen ICI 164,384. This indicates a potential of the coactivator RIP140 to induce a switch of...