Androgen receptor (AR)3 is a hormone-induced transcription factor that controls male sexual development and other important physiologies. Similar to other members of the nuclear receptor family (1, 2), AR has three major functional domains: an N-terminal transactivation domain, a DNA-binding domain, and a C-terminal ligand-binding domain (3-5). Mutations found in each of these domains lead to a series of AR functional defects associated with androgen insensitivity syndrome (AIS) or partial AIS in humans (6, 7). The majority of AIS and partial AIS patients have developmental defects in the male reproductive system. Loss-of-function AR mutations in mice recapitulate many of the reproductive defects found in AIS patients. For example, the AR-deficient (androgen receptor knock-out; ARKO) mouse (8) and the tfm (testicular feminization mutant) mouse (9) both develop severe defects of testicular development and an overall lack of male sexual differentiation, including hypospadias and penile agenesis. The tfm male mouse demonstrates many female secondary structures, including vagina and teats (10).Molecular regulation of AR function can be achieved at several levels, such as spatial-temporal expression of the receptor, modulation of ligand binding, cytoplasm to nucleus translocation, and DNA binding and transcriptional activities (11,12). Prior to hormone binding, steroid receptors form large protein complexes containing the molecular chaperone heat shock protein 90 (Hsp90) as well as various co-chaperone tetratricopeptide repeat (TPR) proteins (13-15). These co-chaperones include Fkbp52 and Fkbp51 (FK506-binding protein 52 and 51, respectively), Cyp40 (cyclophilin 40), and PP5 (protein phosphatase 5). Fkbp52 and Fkbp51 are ubiquitously expressed proteins with peptidyl prolyl cis/trans-isomerase activity that is inhibited by the binding of . Each TPR protein enters into steroid receptor complexes through a direct and competitive binding at the C terminus of Hsp90 via its essential TPR domain (19 -21). Although Fkbp52 and Fkbp51 share a similar domain structure, as well as 60% sequence identity and 75% similarity, they do differ in that Fkbp51 is missing a C-terminal calmodulin-binding domain.To date, most studies on TPR control of the steroid receptor (SR) action have been done using conventional molecular and cellular approaches and using the glucocorticoid (GR) and progesterone (PR) receptors as models. It has been shown that Fkbp52 is localized to both cytoplasm and nucleus but that the cytoplasmic fraction co-localizes with microtubules in a complex containing dynein (22, 23). For these reasons, it was pro-
A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A. M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894 -6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR⅐hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.Unliganded steroid receptors exist in cytosols in heterocomplexes with the abundant, ubiquitous, and essential heat shock protein hsp90 1 (for review, see Ref. 1). The glucocorticoid receptor (GR) must be in heterocomplex with hsp90 for it to have steroid binding activity (2, 3). The ligand binding domain (LBD) is the region of the receptor that interacts with hsp90 (1), and biochemical data (4) coupled with data from GR mutants (5, 6) support the notion (3) that formation of a complex with hsp90 opens up a hydrophobic pocket in the LBD to access by steroid. Steroid receptor⅐hsp90 heterocomplexes are formed in an ATP-dependent process by a multiprotein chaperone system that has been studied most extensively in reticulocyte lysate (7,8) but is present in lysates of both animal and plant cells (9).The receptor⅐hsp90 heterocomplex assembly system has now been reconstituted (10 -14), and five purified proteins, including hsp90, hsp70, 2 Hop (hsp organizer protein), hsp40, and p23, are required for optimally efficient assembly (for review of heterocomplex assembly, see Refs. 15 and 16). Only two of these proteins, hsp70 and hsp90, are absolutely required for opening the steroid binding cleft in the GR LBD, and the other three proteins act as co-chaperones that increase the overall efficiency of GR⅐hsp90 heterocomplex assembly (17).Hop binds independently to hsp90 and hsp70 to form an hsp90⅐Hop⅐hsp70 complex (18). Although Hop is not required for opening of the steroid binding cleft in the GR LBD, it increases the rate of the process (17). The peptide binding activity of hsp70 is coupled to the binding of ADP versus ATP (for review...
Glucocorticoid action in cells is mediated by a specific receptor protein, the glucocorticoid receptor (GR). GR is a member of a superfamily of ligand-inducible transcription factors that control a variety of physiological functions; such as, metabolism, development, and reproduction. Unliganded GR is predominantly localized within the cytoplasm but rapidly and efficiently translocates to the nucleus following hormone binding. This review will focus on the intracellular signaling pathway utilized by the GR including the mechanisms that control its intracellular trafficking, hormone binding and transcriptional regulation. Many receptor-interacting proteins are involved in distinct steps in GR signal transduction, each with a unique mechanism to regulate receptor action and providing potential drug targets for the manipulation of cellular responses to glucocorticoids.
Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membranebased steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.The glucocorticoid receptor (GR) 1 is a ligand-activated transcription factor whose macromolecular structure in the absence of hormone contains heat shock protein 90 (Hsp90) and tetratricopeptide repeat (TPR) proteins. To date, four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) have been found in so-called "mature" GR complexes, i.e., GR that is competent for binding of hormone. Because Hsp90 contains only one binding site for these proteins, four distinct GR heterocomplexes have been identified on the basis of TPR protein content (reviewed in ref 1). Although such heterogeneity implies differential functions for each GR complex, very little is known about this process. In recent work by our laboratory, differential roles for FKBP52 and FKBP51 in the regulation of hormone-induced transport of GR were uncovered (2). It was found that binding of...
We have identified a new first step in the hormonal activation of the glucocorticoid receptor (GR). Rather than causing immediate dissociation of the cytoplasmic GR heterocomplex, binding of hormone-induced substitution of one immunophilin (FKBP51) for another (FKBP52), and concomitant recruitment of the transport protein dynein while leaving Hsp90 unchanged. Immunofluorescence and fractionation revealed hormone-induced translocation of the hormone-generated GR⅐Hsp90⅐FKBP52⅐dynein complex from cytoplasm to nucleus, a step that precedes dissociation of the complex within the nucleus and conversion of GR to the DNAbinding form. Taken as a whole, these studies identify immunophilin interchange as the earliest known event in steroid receptor signaling and provide the first evidence of differential roles for FKBP51 and FKBP52 immunophilins in the control of steroid receptor subcellular localization and transport. The glucocorticoid receptor (GR)1 is a hormone-activated transcription factor that requires hormonally driven movement to its site of action within the nucleus. In the absence of hormone, the GR is recovered in the cytosolic fraction of cells as an oligomeric complex containing one molecule of receptor and two molecules of heat shock protein 90 (Hsp90), to which the receptor binds directly (for review see Ref. 1). An intriguing recent development, however, is that hormone-free receptor is not found as a single, well defined complex but exists as a mixture of complexes. Although all of these complexes contain receptor and Hsp90, each contains only one molecule of either FKBP52, FKBP51, Cyp40, or PP5. The latter proteins have been classified as TPR domain proteins based on the presence of several tetratricopeptide repeat domains that are their sites of interaction with Hsp90 (2, 3). FKBP52, FKBP51, and Cyp40 are also members of the immunophilin family of proteins (4 -6). Thus, it is now clear that up to four distinct receptor heterocomplexes are possible, even within the same cell or tissue; yet almost nothing is known about the differential roles served by each immunophilin in steroid receptor responses.It is generally accepted that the first event in hormonal activation of GR is dissociation of hormone-bound GR from Hsp90 and the TPR proteins, followed by nuclear translocation of the GR and all other downstream events. Hormone-induced dissociation of the complex is a rapid event, occurring both in the intact cell (7) and in cytosolic preparations (8). In cytosols, dissociation of GR complexes has been shown to require warming (typically 20 -25°C) in addition to hormone, and this process can be blocked by molybdate and other transition metal oxyanions (8, 9). Indeed, because of the ability of molybdate to effectively block dissociation, it has been assumed that molybdate-stabilized receptors are more or less "frozen" in their native, untransformed state even when GR is bound with hormone. In this work, we have examined this assumption by measuring the effect of hormone on the immunophilin content of GR hetero...
Glucocorticoid hormones control diverse physiological processes, including metabolism and immunity, by activating the major glucocorticoid receptor (GR) isoform, GRalpha. However, humans express an alternative isoform, human (h)GRbeta, that acts as an inhibitor of hGRalpha to produce a state of glucocorticoid resistance. Indeed, evidence exists that hGRbeta contributes to many diseases and resistance to glucocorticoid hormone therapy. However, rigorous testing of the GRbeta contribution has not been possible, because rodents, especially mice, are not thought to express the beta-isoform. Here, we report expression of GRbeta mRNA and protein in the mouse. The mGRbeta isoform arises from a distinct alternative splicing mechanism utilizing intron 8, rather than exon 9 as in humans. The splicing event produces a form of beta that is similar in structure and functionality to hGRbeta. Mouse (m)GRbeta has a degenerate C-terminal region that is the same size as hGRbeta. Using a variety of newly developed tools, such as a mGRbeta-specific antibody and constructs for overexpression and short hairpin RNA knockdown, we demonstrate that mGRbeta cannot bind dexamethasone agonist, is inhibitory of mGRalpha, and is up-regulated by inflammatory signals. These properties are the same as reported for hGRbeta. Additionally, novel data is presented that mGRbeta is involved in metabolism. When murine tissue culture cells are treated with insulin, no effect on mGRalpha expression was observed, but GRbeta was elevated. In mice subjected to fasting-refeeding, a large increase of GRbeta was seen in the liver, whereas mGRalpha was unchanged. This work uncovers the much-needed rodent model of GRbeta for investigations of physiology and disease.
FK506-binding protein 52 (FKBP52) is a tetratricopeptide repeat protein that associates with steroid receptors in complexes containing heat shock protein 90. To investigate the role of FKBP52 in steroid-regulated physiology, we generated FKBP52-deficient mice. FKBP52 (-/-) females are sterile due to a complete failure of implantation, a process that requires estrogen (ER) and progesterone receptors (PR). Because the uterus expresses two forms of PR, PR-A and PR-B, we investigated all three receptors as potential targets of FKBP52 action. FKBP52 (-/-) uteri showed a normal growth response to estradiol, and unaltered expression of genes controlled by ER and PR-B. In contrast, FKBP52 (-/-) uteri were neither able to express two PR-A-regulated genes, nor undergo decidualization in response to progesterone, suggesting that FKBP52 specifically regulates PR-A at this organ. Analysis of uterine PR heterocomplexes showed preferential association of FKBP52 with PR-A compared with PR-B. Loss of FKBP52 neither disrupted the PR-A/heat shock protein 90 interaction, nor impaired uterine PR-A hormone-binding function, demonstrating the essential role of FKBP52 in PR-A action to be downstream of the hormone-binding event. Transcription studies in +/+ and -/- mouse embryonic fibroblast cells showed a near-complete loss of PR-A activity at mouse mammary tumor virus and synthetic progesterone response element promoters, although partial reductions of ER and PR-B were also observed. Partial disruptions of ovulation and mammary development were also found in FKBP52 (-/-) females. Taken as a whole, our results show FKBP52 to be an essential regulator of PR-A action in the uterus, while being a nonessential but contributory regulator of steroid receptors in the mammary and ovary. These data may now provide the basis for selective targeting of steroid-regulated physiology through tetratricopeptide repeat proteins.
It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.
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