Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Here we show that maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. We propose that this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction.

The orphan nuclear receptors SF-1 and LRH-1 are constitutively active, but it remains uncertain whether their activation is hormone dependent. We report the crystal structure of the LRH-1 ligand binding domain to 2.4 A resolution and find the receptor to be a monomer that adopts an active conformation with a large but empty hydrophobic pocket. Adding bulky side chains into this pocket resulted in full or greater activity suggesting that, while LRH-1 could accommodate potential ligands, these are dispensable for basal activity. Constitutive LRH-1 activity appears to be conferred by a distinct structural element consisting of an extended helix 2 that provides an additional layer to the canonical LBD fold. Mutating the conserved arginine in helix 2 reduced LRH-1 receptor activity and coregulator recruitment, consistent with the partial loss-of-function phenotype exhibited by an analogous SF-1 human mutant. These findings illustrate an alternative structural strategy for nuclear receptor stabilization in the absence of ligand binding.

Despite the fact that many nuclear receptors are ligand dependent, the existence of obligate regulatory ligands is debated for some receptors, including steroidogenic factor 1 (SF-1). Although fortuitously bound bacterial phospholipids were discovered in the structures of the SF-1 ligand-binding domain (LBD), these lipids might serve merely as structural ligands. Thus, we examined whether exogenously added phospholipids would exchange for these bacterial lipids and bind to SF-1. Here, we report the first crystal structure of the SF-1 LBD bound by the exchanged phosphatidylcholine. Although the bound phosphatidylcholine phospholipid mimics the conformation of bound bacterial phosphoplipids, two surface loops, L2-3 and L11-12, surrounding the entrance to the pocket vary significantly between different SF-1 LBD structures. Based on this observation, we hypothesized that a bound ligand might control the conformations of loops L2-3 and L11-12, and that conserved residues in these dynamic loops could influence ligand binding and the receptor function. Consistent with this hypothesis, impaired phospholipid exchange and diminished transcriptional activity were observed for loop L11-12 SF-1 mutants and for the loop L2-3 human mutant R255L. The endocrine disease associated with this L2-3 mutation coupled with our cellular and biochemical data suggest that critical residues at the mouth of the ligand-binding pocket have evolved for efficient binding of phospholipid ligands and for achieving optimal SF-1 activity.

The Dax-1 protein is an enigmatic nuclear receptor that lacks an expected DNA binding domain, yet functions as a potent corepressor of nuclear receptors. Here we report the structure of Dax-1 bound to one of its targets, liver receptor homolog 1 (LRH-1). Unexpectedly, Dax-1 binds to LRH-1 using a new module, a repressor helix built from a family conserved sequence motif, PCFXXLP. Mutations in this repressor helix that are linked with human endocrine disorders dissociate the complex and attenuate Dax-1 function. The structure of the Dax-1:LRH-1 complex provides the molecular mechanism for the function of Dax-1 as a potent transcriptional repressor.Dax-1 ͉ LRH-1 ͉ nuclear receptor ͉ regulation ͉ structure T he orphan nuclear receptor DAX-1/Dax-1 (dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome gene 1) (1) is well known for its role in human pathophysiology. Duplication of the DAX-1 gene causes phenotypic sex reversal in XY individuals (2), and mutations in DAX-1 are responsible for adrenal hypoplasia congenita, an inherited disorder of adrenal gland development (3). During embryogenesis, Dax-1 functions to direct cell differentiation in testes and adrenal tissues (1). In adult physiology, Dax-1 acts as a global repressor of many nuclear receptors, including SF-1, Nur77, ERR␥, ER, AR, PR, and LRH-1 (4-13). Dax-1 also is indispensable to maintaining the pluripotent state of embryonic stem cells (14,15).There is a little information on either the structure or regulatory mechanisms of Dax-1. Dax-1 belongs to a unique family of nuclear receptors (NR0B1) that lack the essential DNA binding domain. Instead, the human Dax-1 N terminus consists of three sequence repeats that include the LXXL/ML motif (''LXXL/ML boxes'' 1-3) (16). This unique N-terminal extension is thought to play a role in subcellular distribution and nuclear localization of . No homologues for the N-terminal region of Dax-1 are known, but its C-terminal domain is a clear homologue of the nuclear receptor ligandbinding domain (LBD) (1). To date, no hormone for Dax-1 has been identified, and the mechanism of its function as corepressor remains under debate (4, 5, 7-9, 12, 18-20). The elucidation of Dax-1 mechanisms has been frustrated by a lack of highresolution structural information. Here we report the first structure of Dax-1 bound to its physiological target, nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2).LRH-1 was first discovered in the liver and intestine, where it regulates genes controlling bile acid synthesis and cholesterol homeostasis (21-24). Recently, LRH-1 was found in human steroidogenic tissues and was shown to activate transcription of genes encoding steroidogenic enzymes (25). In particular, regulation of the CYP19A gene encoding aromatase, which converts androgens to estrogens, gives LRH-1 a pivotal role in estrogen signaling (25-28). Similar to Dax-1, LRH-1 is indispensable to maintaining the pluripotent state of embryonic stem cells (29).Unlike other nuclear receptors that func...