Biochem. J. (2012) 441, 297–303 (Printed in Great Britain) doi:10.1042/BJ20111456 297 BAG-1 diversely affects steroid receptor activity Regina T. KNAPP, Andrea STEINER, Ulrike SCHMIDT, Kathrin HAFNER, Florian HOLSBOER and Theo REIN 1 Max Planck Institute of Psychiatry, Kraepelinstrasse 10, 80804 Munich, Germany Part of the cellular and physiological functions of BAG-1 (Bcl-2- associated athanogene 1) has been ascribed to the ability of this hsp70 (heat-shock protein 70) co-chaperone to regulate steroid receptor activity. BAG-1 has been reported to inhibit the GR (glucocorticoid receptor) and stimulate the androgen receptor, but to leave the activity of the MR (mineralocorticoid receptor) unchanged. Given the high homology between the MR and GR, this disparity in the actions of BAG-1 is surprising. In the present study, we analysed the effect of BAG-1 on the activity of the closely related PR (progesterone receptor). Similarly to the GR, the transcriptional activity of the PR is inhibited by the long and middle isoforms of BAG-1, BAG-1L and BAG-1M, but not by the short isoform, BAG-1S. We found this inhibition to require the hsp70-binding domain of BAG-1. To shed light on the mechanisms that could explain BAG-1’s differential actions on steroid receptors, we tested the binding of BAG- 1M to the PR. Mutational analyses of the PR and BAG-1M revealed that the mode of interaction and BAG-1M-mediated inhibition of the PR differs from the reported scenario for the GR. Surprisingly, we also found binding of BAG-1M to the MR. In addition, BAG-1M was able to inhibit the transcriptional activity of the MR. These data entail a reappraisal of the physiological actions of BAG-1M on steroid receptor activity. Key words: Bcl-2-associated athanogene 1 (BAG-1), glucocorti- coid receptor, mineralocorticoid receptor, progesterone receptor, steroid receptor. INTRODUCTION BAG-1 (Bcl-2-associated athanogene 1) is a co-chaperone of hsp (heat-shock protein) 70 which executes a multitude of molecular, cellular and physiological functions. BAG-1 serves as nucleotide- exchange factor of hsp70 [1] and affects the folding efficiency of this chaperone in a dose-dependent manner [2]. It has been reported to regulate growth and survival of breast cancer cells [3] and is proposed as a marker for the prediction of long-term survival in early-stage cancer [4]. In addition, BAG-1 regulates neuronal differentiation [5] and affects the differentiation and survival of haemopoietic and neuronal cells [6]. Probably related to this, BAG-1 modulates axonal and neurite outgrowth [7]. BAG-1 also plays a role in stress-related diseases such as major depression. Lithium and valproate have been shown to up- regulate BAG-1 expression [8], and mice with genetically altered levels of BAG-1 responded differently in several assays assessing stress-related behaviour [9]. Moreover, chronic mild stress led to down-regulation of BAG-1 expression, which was prevented by concomitant treatment with lithium [10]. It is most likely that several factors contribute to this remarkable plethora of actions. First, BAG-1 is expressed in several isoforms via alternative translation initiation. These isoforms differ in size and are termed accordingly BAG-1L for the long isoform (50 kDa), BAG-1M for the middle isoform (46 kDa) and BAG-1S for the short isoform (33 kDa). These isoforms share an hsp70- interaction domain and therewith their effect on the ATPase activity of hsp70 [1,2]. However, several studies indicate that the isoforms differ in their functions on hsp70-mediated folding and other protein actions [11,12]. The role of BAG-1 as co-chaperone of hsp70 might allow it to influence the many cellular functions of hsp70, which is regarded as a very general chaperone, as opposed to the more specialized chaperone hsp90 [13]. Finally, the range of effects of BAG-1 is amplified by its influence on the activity of a range of proteins that themselves have multifactorial impacts on cell signalling and physiology, for example on the protein kinase raf-1, the retinoblastoma-susceptibility protein Rb1, and steroid receptors [14]. Among the steroid receptors, the GR (glucocorticoid receptor) is inhibited by BAG-1 in an isoform-specific manner [15,16], whereas the highly homologous MR (mineralocorticoid receptor) was reported to act independently of BAG-1 [15]. The function of BAG-1 as specificity determinant of corticosteroid receptor action [17] has potentially important consequences, for example for the balance of activities of the GR and MR which share the same hormone ligand. GR/MR balance plays a crucial role in cerebral stress physiology [18] and BAG-1’s effect on this balance may be related to some of its neurological actions [5–7]. Interestingly, activities of the AR (androgen receptor) and the ER (oestrogen receptor) have been reported to be stimulated by BAG-1L, but not by the shorter isoforms of BAG-1 [19–21], with potential implications for AR- and ER-dependent malignancies [22]. The PR (progesterone receptor) appears to be the only steroid receptor for which an effect of BAG-1 has not yet been established. Homology considerations do not allow us to precisely predict the influence of BAG-1 on PR-mediated transcriptional activity. In the present study, we found PR’s transcriptional activity to be inhibited by BAG-1M. In contrast with the GR, the hinge region of the PR is not required for interaction with and inhibition by BAG-1M. Surprisingly, our analyses also revealed that BAG-1M binds to the MR and inhibits MR-dependent transcription. MATERIALS AND METHODS Cell culture, transfection and reporter gene assays The plasmids MTV-Luc, Gaussia-KDEL, HA-MR, HA-PR, pRK5, BAG-1M and BAG-1Mmut used for the reporter gene assay are as described previously [16,23]. Construction of the Abbreviations used: AR, androgen receptor; BAG-1, Bcl-2-associated athanogene 1; BAG-1L, BAG-1 long isoform; BAG-1M, BAG-1 middle isoform; BAG-1S, BAG-1 short isoform; DBD, DNA-binding domain; ER, oestrogen receptor; GR, glucocorticoid receptor; HA, haemagglutinin; HEK, human embryonic kidney; hsp, heat-shock protein; MR, mineralocorticoid receptor; PR, progesterone receptor; TBS, Tris-buffered saline; WT, wild-type. 1 To whom correspondence should be addressed (email theorein@mpipsykl.mpg.de). c  The Authors Journal compilation c  2012 Biochemical Society www.biochemj.org Biochemical Journal