Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminal ligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as 'orphan receptors' lack identified ligands, however, raising issues about the function of their LBDs. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 A resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a 'classical' binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stable conformation. Our findings highlight a unique structural class of NRs and define a model for ligand-independent NR function.

Androgens, like other steroid hormones, act through intracellular receptors that belong to the superfamily of ligandactivated transcription factors, the nuclear receptors (1, 2). Subsequent to hormone binding, androgen receptor (AR) acquires a new conformation that is capable of interacting not only with specific androgen response elements (AREs) located near or within the promoter regions of regulated genes but also with other transcription-regulating proteins and cofactors. Depending on the physiological context, these interactions promote either activation or repression of specific genes or gene networks that govern the regulation of development, differentiation, and maintenance of male reproductive functions.Transcriptional regulation requires the participation of at least three classes of proteins: (i) proteins that recognize specific DNA motifs, (ii) proteins that are recruited to promoters by protein-protein interactions and act as transcriptional coactivators or corepressors, and (iii) proteins that alter the architecture of chromatin (3). To initiate transcription, nucleotide-sequence-specific factors such as steroid receptors have to communicate with components of the basal transcription apparatus. The mechanisms by which these proteins convey their activating and͞or repressing functions to the basal transcription machinery are not fully understood. Several nuclear receptors have been reported to interact directly with components of the basal transcription apparatus, such as TFIIB, TFIIF, and the TATA box binding protein (4-7). Alternatively, coactivators or corepressors can act as bridging molecules between steroid receptors and general transcription factors (for review, see ref. 8).

The heterotrimeric G protein subunit G s α stimulates cAMP-dependent signaling downstream of G proteincoupled receptors. In this study, we set out to determine the role of G s α signaling in cells of the early osteoblast lineage in vivo by conditionally deleting G s α from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption. Osteoblast number was markedly decreased and osteogenic differentiation was accelerated, resulting in the formation of woven bone. Rapid differentiation of mature osteoblasts into matrixembedded osteocytes likely contributed to depletion of the osteoblast pool. In addition, the number of committed osteoblast progenitors was diminished in both bone marrow stromal cells (BMSCs) and calvarial cells of mutant mice. In the absence of G s α, expression of sclerostin and dickkopf1 (Dkk1), inhibitors of canonical Wnt signaling, was markedly increased; this was accompanied by reduced Wnt signaling in the osteoblast lineage. In summary, we have shown that G s α regulates bone formation by at least two distinct mechanisms: facilitating the commitment of mesenchymal progenitors to the osteoblast lineage in association with enhanced Wnt signaling; and restraining the differentiation of committed osteoblasts to enable production of bone of optimal mass, quality, and strength.

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Ubc9, a homologue of the class E2 ubiquitin-conjugating enzymes, has recently been shown to catalyze conjugation of a small ubiquitin-like molecule-1 (SUMO-1) to a variety of target proteins. SUMO-1 modifications have been implicated in the targeting of proteins to the nuclear envelope and certain intranuclear structures and in converting proteins resistant to ubiquitin-mediated degradation. In the present work, we find that Ubc9 interacts with the androgen receptor (AR), a member of the steroid receptor family of ligand-activated transcription factors. In transiently transfected COS-1 cells, AR-dependent but not basal transcription is enhanced by the coexpression of Ubc9. The N-terminal half of the AR hinge region containing the C-terminal part of the bipartite nuclear localization signal is essential for the interaction with Ubc9. Deletion of this part of the nuclear localization signal, which does not completely prevent the transfer of AR to the nucleus, abolishes the AR-Ubc9 interaction and attenuates the transcriptional response to cotransfected Ubc9. The C93S substitution of Ubc9, which prevents SUMO-1 conjugation by abrogating the formation of a thiolester bond between SUMO-1 and Ubc9, does not influence the capability of Ubc9 to stimulate AR-dependent transactivation, implying that Ubc9 is able to act as an AR coregulator in a fashion independent of its ability to catalyze SUMO-1 conjugation.

Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitro generated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. However, the mechanism by which Nurr1 promotes dopamine cell differentiation has remained unknown. In this study we have used a dopamine-synthesizing cell line (MN9D) with immature characteristics to analyze the function of Nurr1 in dopamine cell development. The results demonstrate that Nurr1 can induce cell cycle arrest and a highly differentiated cell morphology in these cells. These two functions were both mediated through a DNA binding-dependent mechanism that did not require Nurr1 interaction with the heterodimerization partner retinoid X receptor. However, retinoids can promote the differentiation of MN9D cells independently of Nurr1. Importantly, the closely related orphan receptors NGFI-B and Nor1 were also able to induce cell cycle arrest and differentiation. Thus, the growth inhibitory activities of the NGFI-B/Nurr1/ Nor1 orphan receptors, along with their widespread expression patterns both during development and in the adult, suggest a more general role in control of cell proliferation in the developing embryo and in adult tissues.

The orphan nuclear receptor Nurr1 is mainly expressed in the central nervous system but is also detected in certain peripheral tissues such as bone. To elucidate the role of Nurr1 in bone, we examined the ability of Nurr1 to regulate osteopontin (OPN) expression in osteoblastic cell lines. Transfection of Nurr1 in osteoblastic cells increased OPN mRNA expression. A dominant negative Nurr1 variant abolished the ability of PTH to induce OPN expression, suggesting that Nurr1 is involved in mediating the regulation of OPN by PTH. Nurr1 efficiently transactivated a luciferase reporter construct driven by the -857/+191 fragment of the mouse OPN promoter. The activation of the OPN promoter was mediated by the monomeric form of Nurr1, required direct binding of Nurr1 to the OPN promoter, and was dependent on the amino-terminal transactivation function-1. The OPN promoter is also regulated by vitamin D receptor and estrogen-related receptors. We show that Nurr1 and vitamin D activate the OPN promoter in a synergistic fashion, whereas Nurr1-mediated transactivation of the OPN promoter is repressed by estrogen-related receptors. In conclusion, Nurr1 activates the OPN promoter directly in osteoblastic cells, suggesting a role for Nurr1 in the regulation of bone homeostasis.

The small nuclear RING finger protein SNURF is not only a coactivator in steroid receptor-dependent transcription but also activates transcription from steroidindependent promoters. In this work, we show that SNURF, via the RING finger domain, enhances protein binding to Sp1 elements/GC boxes and interacts and cooperates with Sp1 in transcriptional activation. The activation of androgen receptor (AR) function requires regions other than the RING finger of SNURF, and SNURF does not influence binding of AR to cognate DNA elements. The zinc finger region (ZFR) together with the hinge region of AR are sufficient for contacting SNURF. The nuclear localization signal in the boundary between ZFR and the hinge region participates in the association of AR with SNURF, and a receptor mutant lacking the C-terminal part of the bipartite nuclear localization signal shows attenuated response to coexpressed SNURF. Some AR ZFR point mutations observed in patients with partial androgen insensitivity syndrome or male breast cancer impair the interaction of AR with SNURF and also render AR refractory to the transcription-activating effect of SNURF. Collectively, SNURF modulates the transcriptional activities of androgen receptor and Sp1 via different domains, and it may act as a functional link between steroid-and Sp1-regulated transcription.The androgen receptor (AR), 1 a member of the steroid receptor family, acts as a hormone-regulated transcription factor. The N-terminal region contains a powerful ligand-independent transcription activation function-1 (AF-1). The second but weaker activation function (AF-2) localizes to the ligand binding domain; it requires hormone for activation and perhaps also an intramolecular interaction with the AF-1 region (1-6). The apo-ligand binding domains of steroid receptors interact with transcriptional corepressors and heat-shock proteins, and the ligand-induced conformation in the ligand binding domain enables interactions between several coactivators and AF-2 (7-12). The steroid receptor zinc finger region (ZFR) consists of two zinc finger (ZF) structures (13). The first ZF is responsible for contacting the specific hormone response element (HRE), whereas the second ZF stabilizes the receptor-DNA interactions and participates in homodimer formation (13-16). The bipartite nuclear localization signal (NLS) begins immediately C-terminal to the second ZF and continues through the first amino acid residues of the hinge region (17, 18). Besides binding to HREs and mediating the dimerization, the ZFRs of steroid receptors have also other less well characterized but nevertheless important functions. Unlike glucocorticoid receptor (GR) null mice, animals with a GR mutation that prevents dimerization and efficient DNA binding are viable, attesting to the importance in vivo of those GR activities that are independent of DNA binding (19). In human males, mutations in the AR ZFR can lead to either complete or partial androgen insensitivity syndrome (3,20,21). AR ZFR is important in transrepression of AP1-a...