The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 Å crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix α10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

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Constitutive androstane receptor (CAR) induces xenobiotic, bilirubin, and thyroid hormone metabolism as a heterodimer with the retinoid X receptor (RXR). Unlike ligand-dependent nuclear receptors, CAR is constitutively active. Here, we report the heterodimeric structure of the CAR and RXR ligand binding domains (LBDs), which reveals an unusually large dimerization interface and a small CAR ligand binding pocket. Constitutive CAR activity appears to be mediated by the compact nature of the CAR LBD that displays several unique features including a shortened AF2 helix and helix H10, which are linked by a two-turn helix that normally adopts an extended loop in other receptors, and an extended helix H2 that stabilizes the canonical LBD fold by packing tightly against helix H3. These structural observations provide a molecular framework for understanding the atypical transcriptional activation properties of CAR.

Constitutive androstane receptor (CAR) induces xenobiotic, bilirubin, and thyroid hormone metabolism as a heterodimer with the retinoid X receptor (RXR). Unlike ligand-dependent nuclear receptors, CAR is constitutively active. Here, we report the heterodimeric structure of the CAR and RXR ligand binding domains (LBDs), which reveals an unusually large dimerization interface and a small CAR ligand binding pocket. Constitutive CAR activity appears to be mediated by the compact nature of the CAR LBD that displays several unique features including a shortened AF2 helix and helix H10, which are linked by a two-turn helix that normally adopts an extended loop in other receptors, and an extended helix H2 that stabilizes the canonical LBD fold by packing tightly against helix H3. These structural observations provide a molecular framework for understanding the atypical transcriptional activation properties of CAR.

Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligandfree TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligandindependent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.Orphan nuclear receptors constitute a group of nuclear receptors (NRs) 4 that lack known functional ligands (1). Recent evidence has shown that orphan nuclear receptors play key roles in many essential physiological functions and can be used to identify therapeutic targets for human diseases (2-6). In contrast to those of classic hormone receptors, however, the functions of many orphan receptors remain unclear because the investigation of their physiological functions is limited by the lack of information about their structural features and their functional ligands.Testicular receptors form a subgroup of orphan nuclear receptors that consists of two members, TR2 and TR4 (7,8). TR2 and TR4 are expressed in spermatocytes, erythroid cells, liver, and some regions of the brain and are known to play roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system (7,8). TR4 binds to the direct repeat AGGTCA with a variable number of spacer nucleotides (from 0 to 6) in the promoters of putative target genes (9). TR2 and TR4 homodimerize and heterodimerize with each other but do not heterodimerize with retinoid X receptor (RXR) members. The TR2/TR4 heterodimer binds to direct repeats of DNA elements in the embryonic and fetal globin gene promoter and represses globin gene transcription (10, 11).The domain organizations of both TR2 and TR4 are similar to those of many other NR family members. Located in the middle of the receptor is a DNA binding domain (DBD), and mapped to the C terminus is the ligand binding domain (LBD) with a ligand-inducible activation function region, AF-2. Located at the N terminus and upstream of the DBD is the Nterminal domain (NTD), including one or two activation function regions (AF-1), which is con...