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Live cell imaging has revealed the rapid mobility of steroid hormone receptors within nuclei and their dynamic exchange at transcriptionally active target sites. Although a number of other proteins have been shown to be highly mobile within nuclei, the identity of soluble factors responsible for orchestrating nuclear trafficking remains unknown. We have developed a previously undescribed in situ subnuclear trafficking assay that generates transcriptionally active nuclei, which are depleted of soluble factors required for the nuclear mobility of glucocorticoid (GR) and progesterone receptors (PR). Using this system and a fluorescence recovery after photobleaching technique, we demonstrate that nuclear mobility of GR recovered on incubation with reticulocyte lysate was inhibited by geldanamycin, a drug that blocks the chaperone activity of heat-shock protein 90. Direct proof of molecular chaperone involvement in steroid receptor subnuclear trafficking was provided by the ATP-dependent recovery of nuclear mobility of GR and PR on incubation with various combinations of purified chaperone and/or cochaperone proteins. Additionally, for both receptors, the inclusion of hormone during the recovery period leads to a retardation of nuclear mobility. Thus, our results provide a description of soluble nuclear mobility factors and furthermore demonstrate a previously unrecognized role for molecular chaperones in the regulation of steroid receptor function within the nucleus.

Steroid hormone receptors exert much of their effects on cellular physiology through regulating the rate of transcription from unique target genes. Much has been learned about the actions of steroid hormone receptors at regulated promoters through model in vitro studies, but it has always been a challenge to extrapolate these mechanistic insights to molecular events that occur in live cells. However, novel insights have recently been gained regarding the nature of receptor encounters with the transcriptional machinery from elegant experimental approaches that used advances gained in biochemical, molecular biological, cell biological, and biophysical disciplines. Although these is no doubt that steroid hormone receptors represent some of the most mobile proteins within the nucleus, they still maintain their ability to orchestrate a highly ordered recruitment of cofactors and coregulators at specific sites and remain accessible to alternative processing pathways that limit their action. As highlighted in this review, there may be interrelationships between seemingly distinct pathways of receptor trafficking and processing within the nucleus that impact receptor action at regulated promoters.

Steroid hormone receptors interact with several different molecular chaperones. DeFranco and Csermely discuss how the molecular chaperones p23 and Hsp90 may serve to regulate the activity of the ligand-bound steroid receptors within the nucleus. The authors hypothesize that these chaperone proteins may have a proactive role in promoting recycling of receptors once they have interacted with chromatin and in allowing rebinding of ligand once the receptors have been recycled.

The nuclear matrix provides the framework for various processes that occur within the nucleus such as transcription, replication, and splicing. As the composition of the nuclear matrix varies between different cell types, the matrix may influence cell-type-specific gene expression. A number of transcription factors have been shown to be associated with the nuclear matrix, including steroid hormone receptors that were the first transcriptional regulatory proteins localized to this compartment. In this review we highlight the most recent advances in our understanding of steroid hormone receptor targeting to the matrix. A specific nuclear matrix targeting signal (NMTS) has been identified within the glucocorticoid receptor (GR) that includes portions of its DNA-binding domain and tau2 transactivation domains. Distinct nuclear matrix acceptor proteins appear to interact within the GR NMTS and impart divergent effects on the transcriptional regulatory properties of the receptor.

The application of a cell permeabilization technique to the analysis of nuclear import has led to many major breakthroughs in our understanding of this trafficking pathway. Digitonin permeabilization maintains the nucleus in a state competent for faithful, signal-dependent translocation through the nuclear pore complex. This system has also been used to probe the mechanism of hormone-regulated nuclear import through the use of glucocorticoid receptors (GR) as a model substrate. In this report we provide detailed descriptions of the digitonin-permeabilized cell system for use in studies of GR nuclear import. In addition, we present several novel applications that expand the utility of this system to probe for mechanisms of nuclear protein export and subnuclear trafficking., (Copyright 1999 Academic Press.)

Cellular responses to external signals often reflect alterations in gene expression. The activation of cell surface hormone or growth factor receptors upon the binding of appropriate ligands mobilizes signal transduction cascades that can ultimately impact the activity of defined sets of transcription factors. The interpretation of hormonal signals can also be initiated intracellularly, as is the case for steroid hormone receptors. In addition to recognizing specific hormones, steroid hormone receptors also function as transcription factors and directly transduce hormonal signals to activation or repression of unique target genes. The delivery of activated steroid receptors to high-affinity genomic sites must be efficient to account for the rapidity and selectivity of many transcriptional responses to steroid hormones. Thus, the signal transduction capacity of steroid hormone receptors will be affected by the efficiency of receptor trafficking both between different subcellular compartments (i.e., the cytoplasm and nucleus) and within a specific compartment (i.e., the nucleus). This article will highlight the recent advances in our understanding of subcellular and subnuclear trafficking of steroid receptors.

Unliganded steroid receptors exist as heteromeric complexes comprised of heat shock and immunophilin proteins that associate either directly or indirectly with receptor carboxyl-terminal ligand-binding domains. Molecular chaperons, and other proteins associated with steroid receptors, play an important role in the maturation of receptors to a hormone-binding competent state. Steroid receptor-associated 90 and 70 kDa heat shock proteins, hsp90 and hsp70, respectively, have well established roles in protein folding in addition to participating in numerous subcellular trafficking pathways. In this review, we discuss the possible roles that molecular chaperons, such as hsp90, hsp70 and DnaJ proteins, have in steroid receptor trafficking within two distinct subcellular compartments, i.e. the cytoplasm and nucleus.

The synthesis of a number of heat shock proteins is induced in response to various environmental stresses. The resultant induction of heat shock protein gene transcription is brought about by the activation of specific transcription factors termed heat shock factors (HSFs) that exist in a latent form in nonstressed cells. Multiple mechanisms are likely to contribute to negative regulation of HSF activity. One model, which remains controversial, proposes the existence of a negative feedback loop by which one of the products of HSF activation, the 70-kDa heat shock protein (hsp70), acts as one of its negative regulators. Accordingly, HSF activation would proceed upon sequestration of hsp70 by substrates (i.e. unfolded proteins) that may accumulate to relatively high levels in stressed cells. To examine whether putative native substrates of hsp70 (e.g. steroid receptors) could impact the regulation of HSF activity, we have examined whether steroid receptors could activate endogenous HSF. We have found that overexpression of androgen (AR), glucocorticoid (GR), mineralocorticoid, and progesterone receptors in transiently transfected COS-1 cells induced HSF activity. With the exception of AR, which was competent to activate HSF when either liganded or unliganded, all other steroid receptors tested only activated HSF when unliganded. This activity was mapped to the ligand-binding domain of rat GR, making it unlikely that HSF activation results from the induction of a novel gene product by unliganded receptors. As overexpression of hsp70 can eliminate HSF activation by AR, GR, and progesterone receptors, we favor the view that HSF activation can result from the sequestration, by steroid receptor ligand-binding domains, of a negative regulator of HSF, such as hsp70 or an hsp70-associated protein.