A potential specificity determinant of corticosteroid receptor action
Box 3640, D 76021 Karlsruhe, Germany. BAG 1M is a eukaryotic cochaperone that associates with several proteins, including the glucocorticoid receptor (GR). It down regulates GR mediated transactivation by a mechanism that requires its prior recruitment by the liganded receptor from cytoplasm into the nucleus. In the nucleus, it uses a repeated sequence motif ([EEX4]8) at its NH2 terminus to inhibit DNA binding, as well as transactivation functions of the receptor. The mineralocorticoid receptor (MR), a structural and functional homologue of the GR, is unable to translocate BAG 1M into the nucleus, and its transactivation function is also not affected by this protein. This differential regulation of GR and MR activity could be relevant in classic mineralocorticoid tissues such as the kidney in which GR activity needs to be repressed to allow the MR to exert its action. In in situ hybridization studies, we show that BAG 1M is expressed in the kidney. Its expression pattern, especially in the developing kidney,[url=http://www.masterclass-detailing.co.uk/category/ralph-lauren-polo-outlet/]Ralph Lauren Polo Outlet[/url], correlated well with that of the GR. These proteins, particularly hsp90, hsp70, and p60, keep the receptors in a state competent for ligand binding2. Molecular chaperones of the hsp70/hsc70 family play an important role in folding, translocation, and degradation of proteins in eukaryotic cells because of their ability to bind and stabilize non native protein conformation3,4. They recognize and bind extended hydrophobic segments in substrates in a process that allows denatured proteins to be bound and released alternatively to effect protein folding.
Members of the hsp40 and p60/Hop families modulate the protein folding activity of hsp70 through interaction with the amino and carboxy terminal domains of this protein5,6. In this reaction, Hop establishes a physical link between hsp70 and hsp907. A protein termed Hip (hsc70 interacting protein) has been identified as a chaperone cofactor interacting with the ATPase domain of hsc70 and prolonging the interaction of this protein with various target substrates8,9.
Recently,[url=http://www.article-web.co.uk/category/discount-hermes-bags/]Discount Hermes Bags[/url], another member in the family of chaperones and cochaperones has been identified and termed Hap46, BAG 1M, or RAP46. Hap46/RAP46/BAG 1M interacts with the amino terminal adenosine 5' triphosphate (ATP) binding domain of hsp70/hsc7010,11 and competes with Hop as well as Hip for binding to this molecular chaperone6,12. RAP46/BAG 1M also interacts with a number of steroid receptors, including the glucocorticoid receptor (GR)13. We have shown in transient overexpression studies in simian kidney COS 7 cells that BAG 1M distinguishes between the action of the GR and mineralocorticoid receptor (MR)14. Distinguishing between the action of the GR and MR is an important prerequisite for the physiological function of corticosteroids. In vivo,[url=http://www.masterclass-detailing.co.uk/category/polo-outlet/]Polo Outlet[/url], the circulating glucocorticoids bind both the GR and MR with high affinity,[url=http://www.masterclass-detailing.co.uk/category/cheap-polo-shirts/]Cheap Polo Shirts[/url], which prevents access of mineralocorticoid to its own receptor. There is therefore the need for the action of the GR to be inactivated in classic mineralocorticoid targets, which allows the MR to be functionally activated by its ligand. In the kidney or colon, this is achieved by the enzyme 11 hydroxysteroid dehydrogenase, which inactivates glucocorticoids, allowing the mineralocorticoid aldosterone to get to its receptor15,[url=http://www.article-web.co.uk/category/hermes-belts/]Hermes Belts[/url],16. In addition, recent studies suggest that intracellular signaling pathways and certain cellular proteins also distinguish between the action of the GR and the MR17,[url=http://www.article-web.co.uk/category/hermes-wallet/]Hermes Wallet[/url],18.
In this communication,[url=http://www.article-web.co.uk/category/hermes-birkin-bag/]Hermes Birkin Bag[/url], we describe experiments suggesting that the eukaryotic cochaperone BAG 1M contributes to the specificity of action of the MR in classic mineralocorticoid tissues. This was demonstrated in transient transfection studies in COS 7 kidney cells in which BAG 1M down regulated GR activity without any effect on the action of the MR. Furthermore,[url=http://www.article-web.co.uk/category/hermes-online-sale/]Hermes Online sale[/url], in situ hybridization studies in the developing kidney provided additional evidence of coexpression of BAG 1M and the GR but not the MR. Together, these findings strongly implicate BAG 1M in the role of a specificity determinant of corticosteroid receptor action in the kidney.
Top of pageBAG 1 ISOFORMSAt least three isoforms of BAG 1M exist Figure 1. The largest isoform is BAG 1L, which arises through translation initiation at a noncannonical CTG codon upstream and in frame with an ATG used for the synthesis of BAG 1M. In addition, a smaller isoform (BAG 1) exists that arises from a further downstream ATG site19,20. These isoforms are collectively termed BAG 1 proteins in this article. The differently sized proteins are all generated from the same mRNA and arose as a result of the usage of alternative translation initiation sites. All three isoforms contain a ubiquitin like domain (striped box) and an HSP70 binding domain at their carboxy termini. BAG 1L and BAG 1M contain the motif [EEX4]8, while BAG 1 contains two rather than eight copies of this sequence motif. A nuclear localization signal (gray box) is localized at the N terminus of BAG 1L.
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The BAG 1 proteins share a common carboxy terminal sequence through which they interact with hsp70 and its cognate hsc70 to inhibit their ability to renature denatured proteins. The BAG 1 proteins are involved in several other cellular processes. For example, they inhibit apoptosis induced by several agents such as staurosporine21 and growth factor withdrawal22. They also alter the activity of the p53 inducible factor Siah 123 and modulate cell motility of human gastric cancer cells24. In addition, they bind and modulate the activity of several steroid hormone receptors, including the glucocorticoid,[url=http://www.masterclass-detailing.co.uk/category/ralph-lauren-polo/]Ralph Lauren Polo[/url], androgen, and retinoic acid receptors13,14,20,25. Of these,[url=http://www.article-web.co.uk/category/hermes-handbags/]Hermes Handbags[/url], the action of the BAG 1 proteins on the activity of the GR is the most intensively studied13,14 and will therefore be given further attention here.
Top of pageBAG 1M INHIBITS GLUCOCORTICOID RECEPTOR ACTIONBAG 1M has been shown in transient transfection studies in simian kidney cells to down regulate the transactivation function of the GR13. In in vitro glutathione S transferase pull down assay, this protein interacted with the GR through its hinge region (amino acids 491 to 515). This region of the receptor was also shown to contribute to BAG 1M down regulation of the transactivation function of the receptor13. In in vivo laser confocal immunofluorescence experiments, BAG 1M colocalized in the cytoplasm with the GR, as well as with its closely related structural homologue, the MR. Hormone binding by these steroid receptors was, however, not affected by BAG 1M14. Interestingly, BAG 1M was recruited into the nucleus by only the liganded GR, where it down regulated transactivation by this receptor. Transactivation by the MR was not affected by BAG 1M, and neither did this receptor recruit BAG 1M into the nucleus14. This demonstrates a differential regulatory effect of BAG 1M on the action of the GR and MR.
Top of pageDOMAIN MAPPING WITH THE USE OF GR MR CHIMERASTo determine why transactivation by the MR was not affected by BAG 1M, chimeric GR MR constructs were cotransfected into COS 7 simian kidney cells, and their transactivation function was measured in the presence of BAG 1M. The results of this study are summarized in Table 1. BAG 1M had no effect on transactivation by chimeric receptors in which the hinge region and the HBD contain MR sequences such as the constructs MMM and GMM14. No effect of BAG 1M was also observed when either one of these receptor domains contained MR sequences such as in constructs GGM or MMG14. These results show that both the hinge region and the HBD of the GR are required for BAG 1M to exert its negative action on the transactivation function of the GR.
BAG 1M, on the other hand, is prevented from exerting its negative regulatory activities when its last 47 carboxy terminal amino acids are deleted14. Interestingly, deletion of these amino acid sequences also prevented the recruitment of this protein into the nucleus by the GR14, indicating that nuclear localization is essential for the negative regulatory function of BAG 1M. However, nuclear transport alone is not sufficient. When the first 70 NH2 terminal amino acids were deleted, the mutant BAG 1M was transported into the nucleus but was nevertheless unable to inhibit GR action14. The NH2 terminal amino acids of BAG 1M that mediate the negative regulatory function contain the sequence motif [EEX4]8. Partial deletion of this sequence greatly reduced the ability of BAG 1M to inhibit transactivation by the GR14. Two lines of evidence further demonstrated the significance of the [EEX4]8 motif in inhibition of GR mediated transactivation. First, transactivation by the GR was inhibited by BAG 1L, a larger isoform of BAG 1M that also contains the [EEX4]8 motif, but not by the smaller isoform BAG 1 that lacks this sequence14. Second, a fusion protein consisting of the first 70 NH2 terminal amino acids of BAG 1M and an unrelated sequence exerted a strong dominant negative action on transactivation by the GR14. This implicates the possible involvement of a factor that interacts with the [EEX4]8 motif for the down regulation of the transactivation function of the GR.
Top of pageBIOLOGICAL SIGNIFICANCEThe biological significance of the negative regulatory action of BAG 1M is manifold. We have previously shown that BAG 1M inhibition of transactivation by the GR correlated with inhibition of glucocorticoid induced apoptosis13. We also observed that T cells that are resistant to glucocorticoid induced apoptosis express relatively high levels of BAG 1M13. Conversely, conditions that down regulated the endogenous levels of BAG 1M transcripts, such as exposure of thymoma cells to the immunosuppressant rapamycin, enhanced the transactivation potential of the GR and reduced GR mediated apoptosis13. As neoplastic cells generally express relatively high levels of BAG 1M27,28, our results on the inhibition of GR mediated transactivation by this protein may provide mechanistic explanations for the reported cases of glucocorticoid resistance in neoplastic transformation. This point would have to be taken into consideration in studies on glucocorticoid resistance in disorders such as acute lymphocytic and nonlymphocytic leukemia29,30.
Top of pageBAG 1M EXPRESSION DURING DEVELOPMENTBAG 1M down regulation of GR action may also be relevant in the control of GR activity during development. Glucocorticoid levels and GR action need to be controlled during embryogenesis because excess glucocorticoid exposure and action of this hormone are thought to be harmful to the developing fetus, reducing birth weight, and may even predispose to hypertension in adulthood31. The level of glucocorticoid available to the fetus is thought to be controlled by placental 11 hydroxysteroid dehydrogenase type 2. This enzyme inactivates glucocorticoids and protects the developing fetus from high maternal glucocorticoid levels32. Another way whereby glucocorticoid action could be controlled is through down regulation of the action of the GR during development. We therefore examined the pattern of expression of the BAG 1 proteins during different stages of mouse embryogenesis to determine whether it correlated with that of the GR. Using an antibody that detects all the different forms of BAG 1, we identified BAG 1M as the most prominent, if not the only, form of this family of proteins present during different stages of mouse development (results not shown). Based on this finding, we interpreted results we obtained in in situ hybridization studies with a probe that recognizes Bag 1 transcripts to be due to BAG 1M.
BAG 1M was detected as early as 10.5 embryonic days postcoitum (E), with low but above background expression in almost every tissue. However, certain tissues of cartilaginous origin showed exceptionally high levels of expression. These are the branchial arches, fore and hind limbs, Meckel's cartilage, cartilage primordia of the ribs, cartilage rings of the trachea, cartilage primordium of the orbitosphenoid bone as well as noncartilaginous organs such as the thymus and kidney (results not shown). As the kidney is a classic mineralocorticoid target that expresses both the GR and MR, we examined in detail the expression of BAG 1M in this organ.
BAG 1M expression in the kidney was detected by whole mount in situ hybridization as early as E13.5. Between this time and E17.5, BAG 1 was highly expressed throughout all kidney cell types except the glomeruli Figure 2. There was a progressive developmental restriction of expression with time such that by E17.5 Bag 1M transcripts were detected in the convoluted and collecting tubules. Just before birth, no Bag 1M transcripts were detected in the kidney, although they were again detected in the adult kidney. Kidneys at the indicated stages in mouse development were isolated, and whole mount in situ hybridization was performed (A A 657 bp COOH terminal EcoRI NotI restriction fragment of mouse Bag 1 cDNA was subcloned into pBluescript (Stratagene, La Jolla, CA, USA) and was used for in vitro transcription for antisense and sense control digoxygenin labeled RNA probes. The whole mount in situ hybridization experiments were essentially performed as described by Belo et al33. Kidneys from embryos at the stages indicated or adult (12 weeks old) kidney (pp) were isolated, fixed in 4% paraformaldehyde (PFA), and dehydrated, and 7 m paraffin sections were subjected to radioactive in situ hybridization studies (E H'). The same plasmid as indicated earlier in this article was used for the generation of [35S] labeled RNA probes and was used for hybridization as described by Hogan et al34. (A Whole mount in situ hybridization. (E Radioactive in situ hybridizations on tissue sections. (E Bright field illuminations. (E' Corresponding dark field illuminations. (E) Embryonic day postcoitum. Abbreviations are: nb, newborn; pp, 12 weeks postpartum.
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The expression pattern of BAG 1M in the kidney mirrors that of the GR but is different from the expression pattern of the MR. Convincing expression of the MR is observed by E18.5 and postnatally in the renal cortex32, at which time BAG 1M expression is greatly reduced or absent. Since BAG 1M protects cells against apoptosis, it was of interest to compare the expression pattern of this protein with apoptosis in the kidney. Our results on the expression of BAG 1M in the kidney did not correlate in time with apoptosis in this organ. Apoptosis in the developing kidney is detected from E13 to E16, with a gradual decrease by E1835. From E18 to just before birth, the number of apoptotic cells in the kidney was greatly reduced35. It is expected that an antiapoptotic protein such as BAG 1M will show an increased expression at the latter stages of development and just after birth. As the opposite pattern was observed (that is, low expression in the late developmental stages), we suggest that the role of BAG 1M in the developing kidney does not appear to oppose massive apoptosis, but most likely protects against the deleterious effects of a functionally active GR. Knockout studies of this gene will prove the suggested function of BAG 1M in development.
In the adult kidney, where BAG 1, the MR, and GR are all expressed, negative action of BAG 1M on transactivation by the GR but not MR will most likely contribute to the specificity of action of mineralocorticoid in this organ.

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