The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction with and repression of Ad4BP/SF-1. In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP/SF-1 and also with LRH-1 (NR5A2). The target specificity of the LXXLL-related motifs was indicated by the observations that Ad4BP/SF-1, ER␣ (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLLrelated motifs in Dax-1 whereas HNF4 (NR2A1) and ROR␣ (NR1F1) did not. Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by the GAL4 DBD were repressed by Dax-1 to various levels, which correlated with the strength of interaction. Amino acid substitutions revealed that Ad4BP/SF-1 and LRH-1 preferentially interact with L(؉1)XXLL-related motifs containing serine, tyrosine, serine, and threonine at positions ؊2, ؉2, ؉3, and ؉6, respectively. Taken together, our results indicate that the specificities of LXXLL-related motifs in Dax-1 based on their amino acid sequences play an important role in regulation of orphan receptors.More than 70 molecular species of the nuclear receptor superfamily have been identified to date in animals ranging from hydras to humans (47). These transcription factors are generally characterized by the presence of two conserved structural features, a DNA binding domain (DBD) composed of two zinc fingers and a ligand binding domain (LBD) located at the C-terminal region. Various lipophilic ligands interact with the cognate LBDs in apo-type receptors, converting them into an active holo-type conformation that can dynamically regulate transcription (14, 38).The general structure of the LBD is composed predominantly of 12 helices. Interaction with ligand induces allosteric changes in conformation, especially in the configuration of helix 12 at the C terminus of the LBD, leading to transcriptional activation (3, 9) or repression (4, 57). Helix 12 is often referred to as the AF-2 core, which in some receptors is a conserved domain essential for ligand-dependent transcriptional activation (14, 37). Transcriptional coactivators mediate activating signals by binding to nuclear receptors in a ligand-dependent manner (reference 67 and references therein). For this receptor-coactivator interaction, conserved sequences containing a short signature motif of LXXLL (where L is leucine and X can be any amino acid) have been implicated (18,31,60). The conserved leucines in these so-called LXXLL motifs, or NR-boxes, appear to be indispensable for hydrophobic interaction with nuclear receptors (10,46,57). In addition, amino acid residues flanking the core LXXLL are known to influence the binding specificity (8,17,29,36,39,65). In the absence of li...

The results indicate that both PMNs and brain microvessel endothelial cells possess AT1 receptor, that AT1 receptor antagonism ameliorates endothelial injury via inhibition of PMNs and endothelial cell adhesion, and that angiotensin II must be a key factor in hypertensive endothelial injury.

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