The estrogen-related receptors (ERR␣, ERR␤, and ERR␥) form a family of orphan nuclear receptors that share significant amino acid identity with the estrogen receptors, but for which physiologic roles remain largely unknown. By using a peptide sensor assay, we have identified the stilbenes diethylstilbestrol (DES), tamoxifen (TAM), and 4-hydroxytamoxifen (4-OHT) as high-affinity ligands for ERR␥. In direct binding assays, 4-OHT had a Kd value of 35 nM, and both DES and TAM displaced radiolabeled 4-OHT with Ki values of 870 nM. In cell-based assays, 4-OHT binding caused a dissociation of the complex between ERR␥ and the steroid receptor coactivator-1, and led to an inhibition of the constitutive transcriptional activity of ERR␥. ERR␣ did not bind 4-OHT, but replacing a single amino acid predicted to be in the ERR␣ ligand-binding pocket with the corresponding ERR␥ residue allowed high-affinity 4-OHT binding. These results demonstrate the existence of highaffinity ligands for the ERR family of orphan receptors, and identify 4-OHT as a molecule that can regulate the transcriptional activity of ERR␥. N uclear receptors are ligand-activated transcription factors that play critical roles in many aspects of development and adult physiology. Common structural features of nuclear receptors include a central, highly conserved DNA-binding domain (DBD) and a carboxyl-terminal ligand-binding domain (LBD) that contains both a hydrophobic ligand-binding pocket and a transcriptional activation function known as AF-2. Ligand binding induces a conformational change in the LBD, which, in turn, allows the binding of coactivator proteins (1). The binding of coactivators depends on the presence of a short leucine-rich domain with the consensus sequence motif LXXLL (2). Indeed, peptides that contain the LXXLL motif and are as short as eight amino acids can bind to nuclear receptors in a ligand-dependent fashion (3). Many issues concerning the formation and function of nuclear receptor-coactivator complexes remain unresolved, but one emerging theme is that coactivator recruitment brings histone acetyltransferase activity to the transcription complex (4). This activity presumably alters chromatin structure and allows for efficient expression of target genes.The same general concepts of ligand activation and coactivator interaction apply not only to the classical nuclear receptors but also to a growing list of orphan receptors for which natural or synthetic ligands have recently been identified. This list includes the peroxisome proliferator-activated receptors, the liver X receptors, and the farnesoid X receptor, which activate transcription in response to binding fatty acids (5), oxysterols (6, 7), and bile acids (3, 8), respectively. In addition, two other orphan nuclear receptors, the constitutive androstane receptor and the pregnane X receptor, have been shown to regulate the expression of cytochrome-P450 genes in response to a variety of ligands, including the planar hydrocarbon 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), t...

We are developing a system to control G protein signaling in vivo to regulate a broad range of physiologic responses. Our system utilizes G protein-coupled peptide receptors engineered to respond exclusively to synthetic small molecule ligands and not to their natural ligand(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). We have made two prototype RASSLs that are based on the human opioid receptor. Small molecule drugs that activate the receptor are nonaddictive and safe to administer in vivo. Binding and signaling assays reveal 200-2000-fold reductions in the ability of our RASSLs to bind or be activated by dynorphin, an endogenous peptide ligand of the opioid receptor. In a high-throughput signaling assay, these prototype RASSLs expressed in Chinese hamster ovary K1 cells showed little or no response to a panel of 21 opioid peptides but still signaled normally in response to small molecule drugs such as spiradoline. Activation of a RASSL by spiradoline also caused proliferation of rat-1a tissue culture cells. These data provide evidence that G protein-coupled receptors can be made into RASSLs. The potential in vivo applications for RASSLs include the positive enrichment of transfected cells and the development of new animal models of disease.

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