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 pro