Differential Control of Glucocorticoid Receptor Hormone-Binding Function by Tetratricopeptide Repeat (TPR) Proteins and the Immunosuppressive Ligand FK506

Abstract

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...

Essential Role for Co-chaperone Fkbp52 but Not Fkbp51 in Androgen Receptor-mediated Signaling and Physiology

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Arsenic Disruption of Steroid Receptor Gene Activation: Complex Dose−Response Effects Are Shared by Several Steroid Receptors

“…For example, As might alter the activity of trans-membrane transporters such as the P-glycoprotein family of ABC transporters, resulting in fluctuations of the intercellular steroid concentration (27,28). We investigated this possibility by performing whole cell steroid binding assays with [ 3 H]-Dex using a stable line (designated as 10.1.11.14) derived from EDR3 cells (11).…”

Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells

“…FKBP52 association with receptor-Hsp90 complexes results in the enhancement of hormone binding (3,9,10); yet the mechanism by which this occurs is unknown. Although FKBP52 binding to Hsp90 is required for FKBP52 regulation of AR, whether or not FKBP52 interacts directly with the receptor within the context of the chaperone complex is unclear.…”