The glucocorticoid receptor (GR) ␣ interacts with the highly conserved 14-3-3 family proteins. The latter bind phosphorylated serine/threonine residues of "partner" molecules and influence many signal transduction events by altering their subcellular localization and/or protecting them from proteolysis. To examine the physiologic role of 14-3-3 on the glucocorticoid-signaling pathway, we studied the nucleocytoplasmic shuttling and transactivation properties of GR␣ in a cell line replete with or devoid of 14-3-3. We found that endogenous 14-3-3 helped localize green fluorescent proteinfused GR␣ in the cytoplasm in the absence of ligand and potentiated its nuclear export after ligand withdrawal. 14-3-3 also suppressed the transcriptional activity of GR␣ on a glucocorticoid-responsive promoter. Disruption of the classic nuclear export signal of 14-3-3 inactivated its ability to influence the nucleocytoplasmic trafficking and transactivation activity of GR␣, whereas introduction of a mutation inactivating the binding activity of 14-3-3 to some of its partner proteins did not. 14-3-3 bound the ligand-binding domain of GR␣ through its COOH-terminal portion, in a partially liganddependent fashion, while it did not interact with "ligand-binding domain" of GR␤ at all. These results suggest that 14-3-3 functions as a negative regulator in the glucocorticoid signaling pathway, possibly by shifting the subcellular localization/circulation of this receptor toward the cytoplasm through its nuclear export signal. Since 14-3-3 proteins play significant roles in numerous cellular activities, such as cell cycle progression, growth, differentiation, and apoptosis, these actions might indirectly influence the transcriptional activity of GR␣. Conversely, through its 14-3-3 protein interactions, GR␣ may influence these processes. The glucocorticoid receptor (GR)1 belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins and mediates the diverse and pivotal actions of glucocorticoids in the maintenance of resting and stress homeostasis (1). The human GR consists of two highly homologous isoforms, ␣ and ␤, produced by alternative use of exon 9 ␣ or ␤ of the GR gene. GR␣ represents the classic glucocorticoid receptor, which binds glucocorticoids and mediates almost all known glucocorticoid effects, while GR␤ does not bind ligand, has dominant negative activity upon GR␣ and unclear physiologic role(s) (2). GR␣, in the ligand-free condition, resides primarily in the cytoplasm in the form of a hetero-oligomer with several heat shock proteins (hsps) and related molecules (2). Upon hormone binding, GR␣ undergoes a conformational change, dissociates from the hsps and translocates into the nucleus depending on nuclear translocation signals 1 and 2 (3-5). Nuclear translocation signal 1 catalyzes rapid transport of the GR through the nuclear pore, employing the importin-mediated pathway, while nuclear translocation signal 2 contributes to a slower traffic via as yet unknown mechanisms (6).After entering the nucleus, GR␣ binds...

Glucocorticoids regulate many crucial biologic functions through their cytoplasmic/nuclear glucocorticoid receptors (GR). Excess, deficiency, or alteration in tissue sensitivity to glucocorticoids has been associated with major causes of human morbidity and mortality. Brx, a cytoplasmic Rho family guanine nucleotide exchange factor, binds to and influences the activity of several nuclear hormone receptors. We examined the functional and molecular interactions between GR and Brx. The glucocorticoid sensitivity of lymphocytes obtained from mice haplo-insufficient for Brx was significantly decreased. Conversely, GR-mediated transcriptional activity of a glucocorticoid response element (GRE)-mediated glucocorticoid-responsive promoter was enhanced by Brx in a guanine nucleotide exchange factor domain-dependent fashion. Brx interacted with GR, forming a ternary complex with RhoA. In a chromatin immunoprecipitation assay, Brx and RhoA were co-precipitated with GREs only in the presence of ligand-activated GR. Extracellularly administered lysophosphatidic acid, which activates its signaling cascade through a specific membrane GTP-binding protein (G-protein)-coupled receptor in a G-protein ␣ 13 -, Brx-, and RhoA-dependent fashion, enhanced GR transcriptional activity, whereas depletion of endogenous Brx attenuated this effect. These findings suggest that glucocorticoid signaling and, hence, the tissue sensitivity to glucocorticoids, may be coupled to extracellular signals via Brx and small G-proteins. Nuclear Brx might act as a local GRE-GR-transcriptosome activator by mediating the effect of small G-proteins on glucocorticoid-regulated genes.