A New First Step in Activation of Steroid Receptors

Abstract

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...

Hsp90-binding Immunophilins Link p53 to Dynein During p53 Transport to the Nucleus

“…Both of these immunophilins and the immunophilin homolog PP5 have been shown to exist in cytosols in complexes that contain cytoplasmic dynein (29). Here, we use an antibody against the dynein intermediate chain (DIC) to show that dynein is coimmunoadsorbed with both the GR and p53, as previously reported (23,24,30). FKBP51 is another immunophilin that has been found in steroid receptor⅐hsp90 heterocomplexes, where it seems to counter the increase in steroid-binding affinity of the hsp90-bound GR caused by FKBP52 (38).…”

“…GR⅐hsp90 heterocomplexes immunoadsorbed from cell lysates contain cytoplasmic dynein (23,24), a molecular motor that processes along microtubular tracks toward the nucleus (25). These heterocomplexes also contain one of several immunophilins with tetratricopeptide repeat (TPR) domains that interact with a TPR acceptor site on hsp90 (13).…”

Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain

“…In the crystal structure of yeast Hsp90, HM9 and HM10 are in close proximity to each other, suggesting that this transfer might be accomplished without the dissociation of the GR-Hsp90 heterocomplex. Because HM9 is near the binding site of the immunophilins FKBP51 and FKBP52 at the Hsp90 C terminus, transfer of GR from HM10 to HM9 might trigger the observed hormone-induced exchange of the immunophilins FKBP51 by FKBP52 (10). Moreover, the ability of the Hsp90 (22, 29 -30).…”

The Glucocorticoid-induced Gene tdag8 Encodes a Pro-apoptotic G Protein-coupled Receptor Whose Activation Promotes Glucocorticoid-induced Apoptosis

“…Interestingly, several of these genes appear to mediate glucocorticoid resistance. These include the stress response gene dig2, whose expression protects against glucocorticoidinduced apoptosis (38); FKBP51, a known glucocorticoid-regulated component of the glucocorticoid receptor chaperone complex that can mediate glucocorticoid resistance by inhibiting cortisol binding (43)(44)(45); and the ABC transporter 2, which has been identified as a mediator of multidrug resistance (46,47). Also included in this list is bim, a pro-apoptotic member of the bcl-2 family that acts at the mitochondria to activate Bax and release cytochrome c, promoting critical and irreversible steps in the execution phase of the apoptotic process (48,49).…”