The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160, TIF-2/GRIP-1 and CBP/p300 which function as co-activators of transcription, and RIP-140, TIF-1 and TRIP-1/SUG-1 whose functions are unclear. Here we report that a short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. We show that the ability of SRC-1 to bind the oestrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the oestrogen receptor that are required for its ligand-induced activation function. We propose that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.

We show that a number of alkylphenolic compounds, used in a variety of commercial products and found in river water, are estrogenic in fish, birds, and mammals. 4-Octylphenol (OP), 4-nonylphenol, 4-nonylphenoxycarboxylic acid, and 4-nonylphenoldiethoxylate were each capable of stimulating vitellogenin gene expression in trout hepatocytes, gene transcription in transfected cells, and the growth of breast cancer cell lines. The most potent of the chemicals is OP, which was able to stimulate these biological responses to a similar extent as 17 beta-estradiol itself, albeit at a 1000-fold greater concentration. The action of alkylphenols is mediated by the estrogen receptor, as their effects depended on its presence and was blocked by estrogen antagonists. OP, 4-nonylphenol, and 4-nonylphenoxycarboxylic acid appear to possess intrinsic estrogenic activity, because they compete for binding to the estrogen receptor. Moreover, it is likely that they interact with a similar region of the hormone-binding domain as 17 beta-estradiol, because the mutant receptor G-525R, which is defective in estrogen binding, is also insensitive to OP. Like 17 beta-estradiol, OP is capable of stimulating the activity of both transcriptional activation functions, TAF-1 and TAF-2, in the receptor, as judged by analyzing the activity of the wild-type and mutant receptors in transiently transfected cells. The significance of our results will depend to a large extent on the degree of exposure of wildlife and humans to these estrogenic alkylphenolic compounds.

A conserved region in the hormone‐dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti‐estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen receptor in intact cells and modulates its transcriptional activity in the presence of estrogen, but not the anti‐estrogen 4‐hydroxytamoxifen. In view of its widespread expression in mammalian cells, RIP140 may interact with other members of the superfamily of nuclear receptors and thereby act as a potential co‐activator of hormone‐regulated gene transcription.

The estrogen receptor (ER) is expressed in two forms, ER␣ and ER␤. Here we show that ER␣ and ER␤, expressed both in vitro and in vivo, form heterodimers which bind to DNA with an affinity (K d of approximately 2 nM) similar to that of ER␣ and greater than that of ER␤ homodimers. Mutation analysis of the hormone binding domain of ER␣ suggests that the dimerization interface required to form heterodimers with ER␤ is very similar but not identical to that required for homodimer formation. The heterodimer, like the homodimers, are capable of binding the steroid receptor coactivator-1 when bound to DNA and stimulating transcription of a reporter gene in transfected cells. Given the relative expression of ER␣ and ER␤ in tissues and the difference in DNA binding activity between ER␣/ER␤ heterodimers and ER␤ it seems likely that the heterodimer is functionally active in a subset of target cells. Estrogen receptors (ER)1 were recently shown to be encoded by two distinct genes, ER␣ and ER␤ (1, 2). Reverse transcription-polymerase chain reaction (PCR) analysis indicates that ER␤ is highly expressed in prostate and ovary (1, 2), but moderate expression was detected in many other tissues including testis and uterus, some of which also seem to express ER␣ (3). The two receptors which share about 95% homology in the DNA binding domain and 55% homology in the ligand binding domain, both bind to a consensus estrogen response element (ERE) (4) and exhibit similar ligand binding properties (3). They are poorly conserved in the N-terminal domain but ER␤, like ER␣, appears to contain a similar activation domain, activation function 1 (AF-1) sensitive to a mitogen-activated protein kinase pathway (4 -6). In addition, both receptors contain a second activation domain, activation function 2 (AF-2) (7, 8), whose activity is enhanced by the coactivator SRC-1 (4, 9, 10). Thus, although the relative expression of ER␣ and ER␤ varies in cells, their ligand binding, DNA binding, and transactivation properties are rather similar to one another.Steroid hormone receptors usually bind to inverted DNA repeats as homodimers, although the glucocorticoid and mineralocorticoid receptors have been reported to form heterodimers, at least in vitro (11,12). In the classically accepted model of steroid hormone action, the estrogen receptor is sequestered in an inactive state in a multiprotein complex in the absence of hormone (13). Upon estrogen binding, the receptor forms homodimers which then interact with response elements in the vicinity of target genes and modulate rates of gene transcription. In view of the similarity of the ligand binding domain of ER␣ and ER␤ we investigated the possibility that the two receptors may form functional heterodimers in target cells. ER␣ and ER␤ were capable of forming heterodimers on DNA that could bind the coactivator, SRC-1, and appeared to stimulate transcription of a reporter gene. Moreover, we demonstrate that while the region of ER␣ required for homodimerization overlaps with that required for heterodimerization ...

The androgen receptor is unusual among nuclear receptors in that most, if not all, of its activity is mediated via the constitutive activation function in the N terminus. Here we demonstrate that p160 coactivators such as SRC1 (steroid receptor coactivator 1) interact directly with the N terminus in a ligand-independent manner via a conserved glutamine-rich region between residues 1053 and 1123. Although SRC1 is capable of interacting with the ligand-binding domain by means of LXXLL motifs, this interaction is not essential since an SRC1 mutant with no functional LXXLL motifs retains its ability to potentiate androgen receptor activity. In contrast, mutants lacking the glutamine-rich region are inactive, indicating that this region is both necessary and sufficient for recruitment of SRC1 to the androgen receptor. This recruitment is in direct contrast to the recruitment of SRC1 to the estrogen receptor, which requires interaction with the ligand-binding domain.The androgen receptor (AR), a member of the nuclear receptor superfamily (6,45), is a ligand-activated transcription factor with the major ligands testosterone and dihydrotestosterone. It has the overall domain structure common to nuclear receptors, comprising an N-terminal activation domain (activation function 1 [AF1]), a central DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD). A second, ligand-dependent activation function (AF2) in several nuclear receptors, including other steroid hormone receptors, has been well characterized (5,15,19,66), but until recently there was no evidence to support such a function for the AR LBD. Deletion of the LBD results in a molecule with constitutive activity which in many reporter activation assays is equivalent to the maximum activity of the full-length receptor in the presence of ligand, implying that AF1 contributes all the activity of the receptor (35,56,61,82). This finding is in contrast to what occurs with the closely related estrogen receptor (ER), in which AF2 is the major activation domain and AF1 has little independent activity (67). The situation in the AR is still more complex, in that two discrete, overlapping activation domains exist in the N-terminal domain and their usage is context dependent. While almost the entire N terminus (residues 1 to 494) is required for full activity of the full-length receptor, a core that contributes 50% of activity is located between residues 101 and 360, and this region has been termed TAU1. However, in the absence of the LBD a different region, termed TAU5 (residues 370 to 494), mediates activation (34).Upon binding of ligand, steroid hormone receptors adopt an active conformation that facilitates the dissociation of heat shock proteins, dimerization, and binding to response elements in the promoters of responsive genes. These receptors have been shown to interact with some components of the basal transcriptional machinery (3, 9, 10, 32, 46, 59) and also to promote transcription of the gene by interacting with coactivator proteins (23, 68). Coactiva...