Steroid hormone receptors act to regulate specific gene transcription primarily as steroid-specific dimers bound to palindromic DNA response elements. DNA-dependent dimerization contacts mediated between the receptor DNA binding domains stabilize DNA binding. Additionally, some steroid receptors dimerize prior to their arrival on DNA through interactions mediated through the receptor ligand binding domain. In this report, we describe the steroid-induced homomeric interaction of the rat glucocorticoid receptor (GR) in solution in vivo. Our results demonstrate that GR interacts in solution at least as a dimer, and we have delimited this interaction to a novel interface within the hinge region of GR that appears to be both necessary and sufficient for direct binding. Strikingly, we also demonstrate an interaction between GR and the mineralocorticoid receptor in solution in vivo that is dependent on the ligand binding domain of GR alone and is separable from homodimerization of the glucocorticoid receptor. These results indicate that functional interactions between the glucocorticoid and mineralocorticoid receptors in activating specific gene transcription are probably more complex than has been previously appreciated.The effects of corticosteroids are determined through asymmetric distribution of the mineralocorticoid and glucocorticoid nuclear hormone receptors (MR and GR) and the protective effects of 11␤-hydroxysteroid dehydrogenase, which selectively metabolizes glucocorticoids (2, 20, 31). MR is highly sensitive to both mineralocorticoids and glucocorticoids, while GR responds only to higher levels of glucocorticoids and is mostly insensitive to mineralocorticoids.Coordinate signaling by GR and MR is specifically relevant to tissues such as the brain, where an abundance of MR and GR in areas such as the hippocampus is accompanied by an absence of 11␤-hydroxysteroid dehydrogenase (14). Indeed, the effects of GR and MR are critical for homeostatic control of CAl pyramidal neurons, where the two receptors differentially mediate the control of ion regulation and transmitter responsiveness (27). Thus, MR and GR signaling influence memory, mood, and neuronal survival. Elevated cortisol levels correlate with depression and other stress-related psychopathologies and with a long-term attenuation of serotonin signaling (28,29,61).GR and MR function predominantly to regulate specific gene expression patterns through palindromic response elements that accommodate receptor dimers (1). The DNA binding domains (DBDs) of the steroid hormone receptors are highly conserved. As a result, GR and MR, as well as progesterone receptors (PR) and androgen receptor (AR), bind in closely related ways to broadly overlapping response elements. Homodimerization contacts mediated through the receptor DBDs occur on DNA binding and are mediated through specific contacts involving residues in the second zinc finger of the receptor DBDs (38).The potential for transcriptional regulation via heteromeric complexes of these steroid receptors has re...

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naïve MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.

Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.Nuclear hormone receptors are dynamic transcription factors that move rapidly through the nucleus and that, based on the results of heterokaryon fusion assays, are believed to shuttle or exchange rapidly between nucleus and cytoplasm. Shuttling offers the potential for rapid modulation of receptor function in response to cytoplasmic signaling pathways. Nuclear receptors are imported into the nucleus through the karyopherin␣/␤ pathway (1, 2). How nuclear receptor export is accomplished is less clear, although recent reports have suggested an integral role for calreticulin (CRT) 1 (3, 4), a calcium binding protein localized to the lumen of the endoplasmic reticulum (5). The naïve glucocorticoid receptor (GR) is a cytoplasmic protein, which is held in a chaperone complex anchored by hsp90 and containing hsp70, immunophilins, and other factors, including p23, where it is poised to bind ligand (6). Upon ligand binding, the chaperone complex is dissociated, and the receptor moves rapidly to the nucleus to regulate specific gene transcription (7). Within the nucleus, the receptor becomes localized to specific sites but exchanges very rapidly with chromatin and remains highly mobile (8). Ligand binding and transcriptional regulation are transient events, with molecular chaperones also being involved in the disassembly of regulatory complexes (9, 10).Heterokaryon fusion assays have indicated that, while localized to the nucleus, nuclear receptors, including liganded GR, traffic continuously and transiently to the cytoplasm (11-16). Upon withdrawal of steroid, shuttling continues for GR as the receptor reassembles into chaperone complexes and slowly reaccumulates in the cytoplasm over a period of several hours (17)(18)(19). Depending on the cell type, the time req...

The D-site binding protein (DBP) is a member of the proline-and acid-rich (PAR) domain subfamily of basic/ leucine zipper proteins and is involved in transcriptional regulation in the liver. Deletion analysis of the DBP protein was carried out in an effort to define the function of the conserved PAR domain. Internal deletions of the protein, i.e. removing portions of the PAR domain, resulted in a substantial loss in transactivation of a high affinity DBP reporter construct when assayed in Hep G2 cells. These same sequences conferred significant transactivation to GAL4 DNA binding domain fusion proteins, indicating that this region acts as part of an independent activation domain comprised of sequences in both the amino terminus and in the PAR domain of DBP. The coexpression of full-length expression constructs for both DBP and hepatic leukemia factor resulted in a dramatic increase in activation mediated by the GAL4-DBP fusion proteins, suggesting the involvement of a regulated coactivator in this process. DBP transactivation appears to be a p300-dependent process, as a 12 S E1A expression construct disrupted DBP-mediated transactivation, and a p300 expression vector, but not a CREB binding protein vector, was able to restore DBP transactivation. These results suggest that the PAR domain is required for DBP activation, which occurs through a regulated, p300-dependent process.