1 REVIEW

Agonist-bound nuclear receptors: not just targets of coactivators

I Fernandes and J H White1 Department of Physiology, McGill University, McIntyre Medical Sciences Bldg., 3655 Drummond St, Montreal, Quebec H3G 1Y6, Canada 1Department of Medicine, McGill University, McIntyre Medical Sciences Bldg., 3655 Drummond St, Montreal, Quebec H3G 1Y6, Canada

(Requests for offprints should be addressed to J H White; Email: [email protected])

Abstract

Members of the nuclear receptor superfamily of ligand-regulated transcription factors are targets of a wide range of lipophilic signaling molecules as well as several drugs and xenobiotics that modulate many aspects of physiology and metabolism. Agonist binding to receptors is associated with recruitment of coactivators, which are essential for activation of target gene transcription. However, several biochemical and molecular genetic studies have shown that a full understanding of the function of agonist-bound receptors must also accommodate the recruitment of corepressors. These factors may attenuate agonist-induced transactivation, act more transiently as part of a cycle of cofactors recruited to target promoters by ligand-bound receptors, or function in hormone-dependent repression of target gene expression. Journal of Molecular Endocrinology (2003) 31, 1–7

The nuclear receptor superfamily – have broad potential as anticancer agents. To primary targets of lipophilic signaling understand the physiological and pharmacological molecules actions of specific ligands, it is essential to fully characterize the biochemical events induced by Nuclear receptors are ligand-regulated transcrip- their binding to target receptors. tion factors whose activities are controlled by a Nuclear receptors contain well-conserved DNA range of lipophilic extracellular signals, including binding domains (DBD) and ligand binding steroid and thyroid hormones, metabolites of domains (LBD). DBDs control (ligand-dependent) vitamins A (retinoids) and D, cholesterol metabo- recognition by nuclear receptors of specific DNA lites, bile acids, specific prostaglandins, and sequences found in promoters of target genes that xenobiotics (Chawla et al. 2001, McKenna & are known collectively as hormone response O’Malley 2002). Nuclear receptors have been elements (Sanchez et al. 2002). LBDs are located in intensively studied in both academia and the the C-terminus of receptors. Crystal structures of pharmaceutical industry (all 48 members of the agonist- and antagonist-bound LBDs have revealed human superfamily were identified prior to highly conserved
helical structures (Renaud & sequencing of the human genome). Importantly, Moras 2000). Conformational changes induced by they are the targets of numerous drugs, including ligand binding control recruitment of specific synthetic steroid hormone agonists and antagonists, cofactors required for the complex biochemical thiazolidenedione antidiabetics, modifiers of choles- events underlying transcriptional regulation. terol and bile acid metabolism, and analogs of Agonist binding reorients the C-terminal AF-2 vitamins A and D, which, among other actions, helix (helix 12) to create a binding pocket

Journal of Molecular Endocrinology (2003) 31, 1–7 Online version via http://www.endocrinology.org 0952–5041/03/031–1 © 2003 Society for Endocrinology Printed in Great Britain

Downloaded from Bioscientifica.com at 10/01/2021 11:33:54PM via free access 2 I FERNANDES and J H WHITE · Agonist-bound nuclear receptors

recognized by signature motifs of coregulatory boxes (Perissi et al. 1999). Ligand binding induces proteins required for transcriptional regulation. movement of the AF-2 helix and displacement of N-CoR and SMRT. N-CoR or SMRT are components of multiprotein complexes implicated Recruitment of coregulatory proteins in transcriptional repression and histone deacetyl- by nuclear receptors ation. Histone deacetylases (HDACs) are divided into three classes based on homology, domain Numerous coregulatory proteins have been structure, subcellular localization, and catalytic identified that control transcriptional regulation properties (Ng & Bird 2001). NCoR and SMRT by nuclear receptors (Glass & Rosenfeld 2000, are components of several different complexes McKenna & O’Malley 2002). They are classified as containing distinct combinations of ancillary coactivators or corepressors depending on whether proteins and class I or class II HDACs (Rosenfeld they promote or inhibit initiation of target gene & Glass 2001), suggesting that their function transcription. The diversity of coactivators suggests depends on cell type, combinations of transcription that transcriptional activation occurs through factors bound to specific promoters, and phase of recruitment of multiple factors acting sequentially the cell cycle. or in combination. The most extensively charac- terized coactivators are the p160 proteins (Glass & Rosenfeld 2000, McKenna & O’Malley 2002, and Agonist-dependent recruitment of references therein). Many coactivators including corepressors p160 proteins interact with ligand-bound receptors through LXXLL motifs, known as nuclear receptor Hormone binding, particularly by steroid hormone (NR) boxes (Voegel et al. 1996, Heery et al. 1997). receptors, is widely associated with activation of Alpha-helical NR boxes are oriented within a target gene transcription. However, a model of hydrophobic pocket of LBDs containing the receptor action where only coactivators are repositioned AF-2 helix by a charge clamp formed recruited to agonist-bound receptors cannot by conserved residues in helices 3 and 12 (Renaud account fully for nuclear receptor action, or for all & Moras 2000). p160 coactivator binding recruits of the coregulatory proteins identified to date. other factors essential for transactivation, including Several NR box-containing coregulators have been CREB binding protein (CBP) and its homolog identified that function as corepressors or have p300 (Glass & Rosenfeld 2000, McKenna & mixed coactivator–corepressor functions. One of O’Malley 2002). Several coactivators including the first coregulatory proteins identified, transcrip- CBP/p300 and associated factor p/CAF possess tional intermediary protein 1
(TIF1
), is an NR histone acetyltransferase (HAT) activity. HAT box-containing protein that was isolated from a activity essentially caps positively charged lysine two-hybrid screen for ligand-dependent cofactors of residues and loosens their association with DNA, nuclear receptors (Le Douarin et al. 1995). Fusion of facilitating chromatin remodeling and subse- TIF1
to the DBD of GAL4 generates a powerful quent access of the transcriptional machinery to repressor whose activity can be blocked by the promoters. HDAC inhibitor trichostatin A (TSA) (Nielsen et al. The corepressors nuclear receptor corepressor 1999). However, unlike its homolog TIF, TIF1
(N-CoR) and silencing mediator for retinoic acid does not associate strongly with factors such as receptor and thyroid hormone receptor (SMRT) heterochromatin protein 1 (HP1) that are impli- were isolated as factors mediating ligand- cated in heterochromatin formation (Nielsen et al. independent repression by thyroid and retinoic acid 1999). A similar two-hybrid screen yielded nuclear receptors (Chen & Evans 1995, Horlein et al. 1995). receptor-binding SET domain-containing protein Unlike p160 proteins, N-CoR and SMRT do not (NSD1), a 2588 amino acid protein that contains interact with LBDs when the AF-2 helix is in the both NR boxes and LXXI/HIXXXI/L motifs agonist-bound conformation. Rather, they recog- similar to those in N-CoR and SMRT. These nize LBDs in a hormone-free or, in some cases, motifs control its interaction with multiple nuclear antagonist-bound conformation through LXXI/ receptors in either a ligand-independent or HIXXXI/L motifs that resemble extended NR –dependent fashion (Huang et al. 1998). NSD1 also

Journal of Molecular Endocrinology (2003) 31, 1–7 www.endocrinology.org

Downloaded from Bioscientifica.com at 10/01/2021 11:33:54PM via free access Agonist-bound nuclear receptors · I FERNANDES and J H WHITE 3

Figure 1 Schematic representation of the primary structures of RIP140 and LCoR. The NR boxes of RIP140 and the LCoR NR box are represented by black bars. Positions of CtBP binding motifs are indicated by white boxes. HDAC binding domains are overlined. See text for details.

contains several distinct coactivation or corepres- LCoR and RIP140 are molecular sion domains, raising the possibility that their scaffolds for several repressors of functions may be selectively modulated by second- transcription ary signal transduction pathways, thus controlling whether NSD1 acts as a coactivator or a LCoR and RIP140 recruit similar cofactors, reveal- corepressor. ing remarkable parallels in their mechanisms of Receptor interacting protein of 140 kDa action in spite of very limited homology (Fig. 1). (RIP140) was initially characterized as a coactivator Corepression by LCoR and RIP140 can be blocked (Cavaillès et al. 1995) that interacted with by the HDAC inhibitor TSA, and both LCoR and agonist-bound receptors through multiple NR RIP140 interact directly with HDACs. LCoR was boxes (Heery et al. 1997). However, subsequent found to interact directly with HDACs 3 and 6, but work showed that RIP140 functioned as a not HDACs 1 and 4 in vitro, and endogenous LCoR corepressor that competes with p160s for binding coimmunoprecipitated with HDACs 3 and 6 to agonist-bound LBDs, blocking coactivation (Fernandes et al. 2003). Mutagenesis studies have in vivo (Eng et al. 1998, Lee et al. 1998, Miyata et al. shown that HDACs 3 and 6 interact with distinct 1998, Treuter et al. 1998). Recently, a ligand- domains of LCoR (Fig. 1 and our unpublished dependent corepressor, LCoR, was identified in a results). Similarly, RIP140 has been shown to inter- screen for proteins that interacted with the estrogen act directly with HDACs 1 and 3 (Wei et al. 2000). receptor
(ER
) LBD in an estradiol-dependent RIP140 is also a target of C-terminal binding manner (Fernandes et al. 2003). LCoR corepressed protein (CtBP) corepressors (Vo et al. 2001), and ligand-dependent transactivation of several recep- mutation of the CtBP binding motif of RIP140 tors and functioned as a repressor when fused to severely attenuates its corepressor function. CtBP1 the GAL4 DBD. LCoR contains a single LXXLL was originally identified as a factor that interacted motif and, like RIP140, interacts with a number of with the C-terminus of the adenoviral oncoprotein receptors in the presence of agonist, but not E1A, and mutations in the CtBP binding motif of antagonist. LCoR binds to the same coactivator- E1A increase its oncogenicity (Chinnadurai 2002). binding pocket as p160s (Fernandes et al. 2003). Remarkably, sequence analysis of LCoR revealed However, mutagenesis studies have suggested tandem motifs PLDLTVR and VLDLSTK (Fig. 1) that LCoR recognizes a distinct and extended that are homologous to the consensus P/VLDLS/ portion of helix 3, suggesting that it may be TXK/R defined as a binding site for CtBPs (Vo possible to develop synthetic agonists that promote et al. 2001). In vitro binding studies and coimmuno- recruitment of LCoR and not p160 proteins. precipitations revealed that the CtBP binding is www.endocrinology.org Journal of Molecular Endocrinology (2003) 31, 1–7

Downloaded from Bioscientifica.com at 10/01/2021 11:33:54PM via free access 4 I FERNANDES and J H WHITE · Agonist-bound nuclear receptors

disrupted only upon mutation of both sites protein–DNA interactions from those associated (Fernandes et al. 2003, and our unpublished results). with activation, and include hormone-dependent Immunocytochemical studies revealed a substantial recruitment of corepressors (Fig. 2A). This notion is overlap of CtBPs and LCoR in discrete nuclear supported by studies of the recently character- bodies. ized DEAD box RNA helicase DP97, which The binding of CtBPs may explain the has corepressor activity and is recruited to ER
in observation that the sensitivity of corepression by the presence of estradiol (Rajendran et al. 2003). LCoR to the HDAC inhibitor TSA is receptor Knockdown of its expression is associated with dependent. While inhibition of estrogen- and attenuation of the repression of genes inhibited by glucocorticoid-dependent transcription was TSA hormone-bound ER
. sensitive, corepression of transactivation by recep- Alternatively, ligand-dependent corepressors may tors for progesterone (PR) or vitamin D, or be regulated by signals that act to attenuate repression by GAL–LCoR fusions was largely TSA hormone-dependent transactivation. Several exper- resistant (Fernandes et al. 2003). Moreover, while iments have shown that nuclear receptor function is corepression of ER
was partially disrupted by modulated by signals other than ligand binding mutation of the CtBP motifs of LCoR, corepression (McKenna & O’Malley 2002). For example, there of PR function was completely blocked by the same are numerous examples of the effects of phos- mutations. Like LCoR, the sensitivity of repression phorylation on nuclear receptor function (Shao & by CtBPs to TSA is dependent on the promoter Lazar 1999). MAP kinase signaling stimulates ER
tested, indicative of HDAC-dependent and function by phosphorylation of Ser118 (Kato et al. -independent modes of action (Chinnadurai 2002). 1995), and coactivation of ER
by p160 amplified in breast cancer 1 (AIB1) is enhanced by MAP kinase phosphorylation of an AIB1 activation Potential roles of hormone-dependent domain, which enhances recruitment of p300 and corepressors in nuclear receptor associated HAT activity (Font de Mora & Brown function 2000). Therefore, there may exist signals that counteract the effects of MAP kinase signaling on While an impressive amount of work has been done ER
function and stimulate the recruitment to identify nuclear receptor coactivators and co- and/or activity of LCoR, RIP140 or other repressors, many important questions remain to be corepressors, thus attenuating receptor activity. answered. First of all, it remains unclear which These may simply alter the balance between the signals determine whether an agonist-bound recep- activities of MAP kinases and MAP kinase tor will recruit an NR box containing coactivator or phosphatases, or there may be independent corepressor. In addition, we still do not understand signaling events that enhance corepressor function the roles of hormone-dependent recruitment of to the detriment of coactivation. Changes in the corepressors in regulating nuclear receptor function. activities of these pathways (Fig. 2B) would thus Distinct, non-mutually exclusive models of action modulate competition between ligand-dependent can be proposed (Fig. 2). Corepressors may be coactivators and corepressors for hormone-bound selectively recruited during hormone-dependent receptors over relatively long-term periods (several repression of gene transcription (Fig. 2A). Steroid cycles of transcriptional initiation). hormone and other nuclear receptors bound to It is also possible that ligand-dependent co- response elements have been widely shown to func- repressors may intervene more rapidly and tion as hormone-dependent transcriptional activa- transiently to control transcription (Fig. 2C). tors, and recruit coactivators. However, recent Current models suggest that multiple factors microarray studies of hormone-regulated gene required for initiation of transcription are recruited expression have emphasized the fact that receptors by transcription factors sequentially. ER
and also repress transcription of some target genes, with p160s associate with promoters within minutes of profiles of regulation mirroring those of activated addition of hormone (Shang et al. 2000, Burakov genes (Inoue et al. 2002, Lin et al. 2002, Lobenhofer et al. 2002). Strikingly, studies with fluorescent- et al. 2002, Wan & Nordeen 2002). Target gene tagged receptors have shown that in vivo, even in repression may entail distinct protein–protein or the continuous presence of hormone, steroid

Journal of Molecular Endocrinology (2003) 31, 1–7 www.endocrinology.org

Downloaded from Bioscientifica.com at 10/01/2021 11:33:54PM via free access Agonist-bound nuclear receptors · I FERNANDES and J H WHITE 5

Figure 2 Potential roles of NR box-containing corepressors in hormone-dependent transcription by nuclear receptors. (A) Selective recruitment of corepressors to target genes whose expression is repressed in the presence of hormone. Note that the schematic representation implies direct receptor DNA binding to target promoters. Repression could also occur through receptor–protein interactions with factors bound to the promoter. (B) Inhibition of hormone-dependent transcription due to a change in signaling environment that promotes receptor–corepressor complex binding to a target promoter over receptor– coactivator complex binding. (C) Determination of the degree of hormone-induced target gene expression over the short term due to rapid exchange of receptor–coregulator complexes in a constant signaling environment. CoA, coactivator; CoR, corepressor; H, hormone; S, signal; *, post-translational modification. receptors and recruited factors cycle rapidly (on the et al. 2002). Thus, the relative proportion of order of minutes) on and off target promoters hormone-bound receptors complexed with coacti- (McNally et al. 2000, Stenoien et al. 2001, Becker vators or corepressors at a given time may www.endocrinology.org Journal of Molecular Endocrinology (2003) 31, 1–7

Downloaded from Bioscientifica.com at 10/01/2021 11:33:54PM via free access 6 I FERNANDES and J H WHITE · Agonist-bound nuclear receptors

determine the degree to which target gene Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK & Rosenfeld MG transcription is induced. It will therefore be 1995 Ligand-independent repression by the thyroid hormone important to analyze corepressor recruitment to receptor mediated by a nuclear receptor co-repressor. Nature 377 hormone-responsive promoters by chromatin 397–404. Huang NW, vom Baur E, Garnier JM, Lerouge T, Vonesch JL, immunoprecipitation (ChIP) assays to determine LutzY,ChambonP&Losson R 1998 Two distinct nuclear when they intervene during hormone-dependent receptor interaction domains in NSD1, a novel SET protein that transcription. Clearly much work remains to be exhibits characteristics of both corepressors and coactivators. done to characterize the roles of NR box- EMBO Journal 17 3398–3412. Inoue A, Yoshida N, Omoto Y, Oguchi S, Yamori T, Kiyama R & containing corepressors in controlling nuclear Hayashi S 200 Development of cDNA microarray for expression receptor signaling. However, with the powerful profiling of estrogen-responsive genes. Journal of Molecular molecular and genetic tools available today, the Endocrinology 29 175–192. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchyama S, Sasaki H, next few years should see rapid advances in our Masushige S, Gotoh Y, Hishida E, Kawashima H, Metzger D & understanding of their function. Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270 1491–1494. Le Douarin B, Zechel C, Garnier JM, Lutz Y, Tora L, Pierrat P, Acknowledgement Heery D, Gronemeyer H, Chambon P & Losson R 1995 The N-terminal part of TIF1, a putative mediator of the This work was supported by an operating grant ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO Journal 14 (MT-11704) from the Canadian Institutes of 2020–2033. Health Research. Lee CH, ChinpaisalC&WeiLN1998Cloning and characterization of mouse RIP140, a corepressor for nuclear orphan receptor TR2. Molecular and Cellular Biology 18 6745–6755. References Lin R, Nagai Y, Sladek R, Bastien Y, Ho J, Petrecca K, Sotiropoulou G, Diamandis EP, HudsonT&White JH 2002 Becker M, Baumann C, John S, Walker DA, Vigneron M, McNally Expression profiling in squamous carcinoma cells reveals JG & Hager GL 2002 Dynamic behavior of transcription factors pleiotropic effects of vitamin D3 signaling on cell proliferation, on a natural promoter in living cells. EMBO Reports 3 1188–1194. differentiation and immune system regulation. Molecular Burakov D, Crofts LA, Chang CPB & Freedman LP 2002 Endocrinology 16 1243–1256. Reciprocal recruitment of DRIP/mediator and p160 coactivator Lobenhofer EK, Bennett L, Cable PL, Li LP, Bushel PR & Afshari complexes in vivo by estrogen receptor. Journal of Biological Chemistry CA 2002 Regulation of DNA replication fork genes by 17 beta- 277 14359–14362. estradiol. Molecular Endocrinology 16 1215–1229. Cavaillès V, Dauvois S, L’Horset F, Lopez G, Hoare S, Kushner PJ McKenna NJ & O’Malley BW 2002 Combinatorial control of gene & Parker MG 1995 Nuclear factor RIP140 modulates expression by nuclear receptors and coregulators. Cell 108 transcriptional activation by the estrogen receptor. EMBO Journal 465–474. 14 3741–3751. McNally JG, Muller WG, Walker D, WolfordR&Hager GL 2000 Chawla A, Repa J, Evans RM & Mangelsdorf DJ 2001 Nuclear The glucocorticoid receptor: rapid exchange with regulatory sites receptors and lipid physiology: opening the X-files. Science 294 in living cells. Science 287 1262–1265. 1866–1870. Miyata KS, McCaw SE, Meertens LM, Patel HV, Rachubinski RA Chen JD & Evans RM 1995 A transcriptional co-repressor that & Capone JP 1998 Receptor-interacting protein 140 interacts with interacts with nuclear hormone receptors. Nature 377 454–457. and inhibits transactivation by peroxisome proliferator-activated Chinnadurai G 2002 CtBP, an unconventional transcriptional receptor alpha and liver-X-receptor alpha. Molecular and Cellular corepressor in development and oncogenesis. Molecular Cell 9 Endocrinology 146 69–76. 213–224. Ng HH & Bird A 2001 Histone deacetylases: silencers for hire. Eng FCS, Barsalou A, Akutsu N, Mercier I, Zechel C, Mader S & Trends in Biochemical Science 25 121–126. White JH 1998 Different classes of coactivators recognize distinct Nielsen AL, Ortiz JA, You J, Oulad-Abdelghani M, Khechumian R, but overlapping sites on the estrogen receptor ligand binding Gansmuller A, ChambonP&Losson R 1999 Interaction with domain. Journal of Biological Chemistry 273 28371–28377. members of the heterochromatin protein 1 (HP1) family and Fernandes I, Bastien Y, Wai T, Nygard K, Lin R, Cormier O, Lee histone deacetylation are differentially involved in transcriptional HS, Eng F, Bertos NR, Pelletier N et al. 2003 Ligand-dependent silencing by members of the TIF1 family. EMBO Journal 18 corepressor LCoR functions by histone deacetylase-dependent and 6385–6395. -independent mechanisms. Molecular Cell 11 139–150. Perissi V, Staszewski LM, McInerney EM, Kurokawa R, Krones A, Font de MoraJ&BrownM2000AIB1isasignaling conduit for Rose DW, Lambert MH, Milburn MV, Glass CK & Rosenfeld kinase-mediated growth factor signaling to the estrogen receptor. MG 1999 Molecular determinants of nuclear receptor–corepressor Molecular and Cellular Biology 20 5041–5047. interaction. Genes and Development 13 3198–3208. Glass CK & Rosenfeld MG 2000 The coregulator exchange in Rajendran RR, Nye AC, Frasor J, Balsara RD, Martini PGV & transcriptional functions of nuclear receptors. Genes and Development Katzenellenbogen BS 2003 Regulation of nuclear receptor 14 121–141. transcriptional activity by a novel DEAD box RNA helicase Heery DM, Kalkhoven E, HoareS&Parker MG 1997 A signature (DP97). Journal of Biological Chemistry 278 4628–4638. motif in transcriptional co-activators mediates binding to nuclear Renaud JP & Moras D 2000 Structural studies on nuclear receptors. receptors. Nature 387 733–736. Cell and Molecular Life Science 57 1748–1769.

Journal of Molecular Endocrinology (2003) 31, 1–7 www.endocrinology.org

Downloaded from Bioscientifica.com at 10/01/2021 11:33:54PM via free access Agonist-bound nuclear receptors · I FERNANDES and J H WHITE 7

Rosenfeld MG & Glass CK 2001 Coregulator codes of VoN,FjeldC&GoodmanRH2001 Acetylation of nuclear transcriptional regulation by nuclear receptors. Journal of Biological hormone receptor-interacting protein RIP140 regulates its Chemistry 276 36865–36868. interaction with CtBP. Molecular and Cellular Biology 21 6181–6188. Sanchez R, Nguyen D, Rocha W, White JH & Mader S 2002 Voegel JJ, Heine MJS, Zechel C & Chambon P 1996 The Diversity in the mechanisms of gene regulation by estrogen coactivator TIF2 contains three nuclear receptor-binding receptors. BioEssays 24 244–254. motifs and mediates transactivation through CBP Shang Y, Xiao H, DiRenzo J, Lazar MA & Brown M 2000 binding-dependent and -independent pathways. EMBO Journal 13 Cofactor dynamics and sufficiency in estrogen receptor-regulated 3667–3675. transcription. Cell 103 843–852. Wan YH & Nordeen SK 2002 Overlapping but distinct gene ShaoD&Lazar M 1999 Modulating nuclear receptor function: may regulation profiles by glucocorticoids and progestins in human the phos be with you. Journal of Clinical Investigation 103 breast cancer cell. Molecular Endocrinology 16 1204–1214. 1617–1618. WeiLN,HuXL,ChandraD,SetoE&Farooqui M 2000 Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M, Receptor-interacting protein 140 directly recruits histone O’Malley BW, Smith CL, Belmont AS & Mancini MA 2001 deacetylases for gene silencing. Journal of Biological Chemistry 275 Ligand-mediated assembly and real-time cellular dynamics of 40782–40787. estrogen receptor
–coactivator complexes in living cells. Molecular and Cellular Biology 21 4404–4412. Treuter E, Albrektsen T, Johansson L, Leers J & Gustafsson JA 1998 A regulatory role for RIP140 in nuclear receptor activation. Received 2 February 2003 Molecular Endocrinology 12 864–881. Accepted 8 May 2003

www.endocrinology.org Journal of Molecular Endocrinology (2003) 31, 1–7

Downloaded from Bioscientifica.com at 10/01/2021 11:33:54PM via free access