The hsp90-FKBP52 Complex Links the Mineralocorticoid Receptor to Motor Proteins and Persists Bound to the Receptor in Early Nuclear Events

Abstract

In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm.The mineralocorticoid receptor (MR) is a member of the steroid/thyroid superfamily of nuclear receptors whose transcriptional activity is triggered by aldosterone binding under normal physiologic conditions. Polarized epithelial tissues such as the distal nephron and colon are considered the classical targets of mineralocorticoids to control salt-water balance by induction of sodium reabsorption and thereby regulation of extracellular fluid volume and blood pressure. MR expression and function also extend to nonepithelial cells, such as hippocampal and hypothalamic neurons, cardiomyocytes, vascular endothelium, and adipocytes (for recent reviews, see references 65 and 52 and references therein).MR shares considerable homology with the glucocorticoid receptor (GR), which is exemplified by the ability of some glucocorticoids to bind both receptors. It is now well established (45) that the GR (the best-studied member of the family) forms heterocomplexes with the 90-kDa and 70-kDa heat shock proteins (hsp90 and hsp70, respectively), the acidic protein p23, and proteins that possess sequences of 34 amino acids repeated in tandems, the tetratricopeptide repeat (TPR) proteins. Some of these hsp90-binding TPR proteins have peptidylprolyl-isomerase activity and are intracellular receptors for immunosuppressant drugs such as FK506, rapamycin, and cyclosporine. They belong to the relatively conserved large family of proteins known as immunophilins (IMMs) (48). Among the members of this family, some IMMs have been recovered in steroid receptor-hsp90 complexes, i.e., FKBP52, FKBP51, CyP40, and three IMM-like proteins, protein phosphatase 5 (PP5), XAP2/ARA9, and WISp39 (33, 44). Even though th...

The Emerging Role of TPR-Domain Immunophilins in the Mechanism of Action of Steroid Receptors

“…after the overexpression of the p50/dynactin-2 subunit of dynactin [36]. In all these cases, the nuclear localization of the receptor was not fully inhibited, but it was only impaired (Figure 2-B).…”

“…This suggests the existence of two mechanisms of transport, a rapid Hsp90•FKBP52•dynein complex-dependent mechanism ( Figure 2-B, blue continuous line) and an alternative, slower and heterocomplexindependent mechanism ( Figure 2B, red dotted line), perhaps due to simple diffusion. Importantly, when the nuclear translocation rate of these receptors is impaired, they are highly sensitive to proteasomal degradation [20,36]. The same Hsp90•FKBP52•dynein complex constitutes the molecular machinery responsible for the retrotransport of the proapoptotic factor p53 [65], suggesting that it may play a general role in the retrotransport mechanism of a number of Hsp90-associated factors.…”

“…Even though the biological function of these IMMs remains poorly understood, it is accepted that they are not related to immunosuppression, a property that concerns to the smallest members of the family, CyPA and FKBP12 (see [48] for a recent update). Importantly, it was demonstrated that the intermediate chain of the motor protein dynein coimmunoprecipitates with the Hsp90•FKBP52 complex bound to the GR [21,61,63] and MR [36] suggesting that the motor protein powers the retrograde movement of the receptor. Actually, the use of inhibitors of the ATPase activity of dynein and/or the disruption of the complex impairs the retrotransport of the receptor [36,64].…”

“…Importantly, it was demonstrated that the intermediate chain of the motor protein dynein coimmunoprecipitates with the Hsp90•FKBP52 complex bound to the GR [21,61,63] and MR [36] suggesting that the motor protein powers the retrograde movement of the receptor. Actually, the use of inhibitors of the ATPase activity of dynein and/or the disruption of the complex impairs the retrotransport of the receptor [36,64]. and Hsp70 (and their associated cochaperone Hsp40) are assembled thanks to the presence of Hop/p60 (heat-shock organizing protein, formerly called p60).…”

“…Regardless of their primary localization, however, these receptors (and other nuclear factors) undergo a permanent and dynamic nucleocytoplasmic shuttling. Clearly, such diversity is most likely related to a dissimilar import/export balance, which could be due to the expression balance of TPR-domain immunophilins [36,37], as we will discuss later in this article. The biological relevance of the nucleocytoplasmic shuttling is implied by the fact that the intersection of the predicted interactome for the Hsp90/Hsp70 chaperone machinery and the interactome of steroid receptors represents˜20% of genes whose products are related to intracellular transport and/or nucleocytoplasmic shuttling [38].…”