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.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ER␣) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ER␣-regulated gene expression involves interactions with cointegrators (e.g. p300/ CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ER␣ is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ER␣ at lysine residues within the ER␣ hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ER␣ hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ER␣ acetylation normally suppresses ligand sensitivity. These ER␣ lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ER␣ acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

We find that tamoxifen is a potent activator of estrogen receptor (ER)- mediated induction of promoters regulated by AP-1 sites including the human collagenase gene promoter and constructs in which an AP-1 site is fused to the herpes thymidine kinase promoter. This contrasts with the inability of tamoxifen to activate otherwise identical promoters bearing classical estrogen response elements. Tamoxifen agonism at AP-1 sites is cell type specific, occurring in cell lines of uterine, but not of breast, origin. It thus parallels tamoxifen agonism in vivo. AP-1 proteins such as Jun or Jun/Fos are needed for tamoxifen stimulation, and tamoxifen increases the transcriptional efficiency of these proteins even when they are provided at optimal amounts. The DNA binding domain (DBD) of ER is required for tamoxifen activation at AP-1 sites. In contrast, estrogen activation is partially independent of this domain. This suggests the existence of two pathways of ER action at AP-1: an alpha (DBD-dependent) pathway activated by tamoxifen, and a beta (DBD-independent) pathway activated by estrogen. Fusing VP16 transcriptional activation functions to ER potentiates the beta, but not the alpha, pathway. We discuss models for the two pathways and the possibility that the AP-1 pathway is a major route by which ER affects target tissue growth and differentiation in vivo.

Estrogen receptors (ERs alpha and beta) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ERalpha AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ERalpha utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ERalpha AF-1 and weakly inhibited by AF-2. Indeed, ERalpha truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERbeta, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.

Promoter-bound steroid receptors activate gene expression by recruiting members of the p160 family of coactivators. Many steroid receptors, most notably the progesterone and estrogen receptors, are regulated both by cognate hormone and independently by growth factors. Here we show that epidermal growth factor regulates the activities of the p160 GRIP1 through the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases. ERKs phosphorylate GRIP1 at a specific site, Ser-736, the integrity of which is required for full growth factor induction of GRIP1 transcriptional activation and coactivator function. We propose that growth factors signal to nuclear receptors in part by targeting the p160 coactivators. Nuclear receptors such as the estrogen receptor (ER)1 and progesterone receptor (PR) tether via their DNA binding domain to response elements in the promoter region of target genes and stimulate transcription. To do so the receptors must bind to coactivators that they recruit through transcriptional activation functions, the constitutive AF-1, found in the aminoterminal receptor domain, and the hormone-activated AF-2 in the carboxyl ligand binding domain (LBD) (for review, see Ref. 1). Perhaps the most important of these coactivators is the p160 family, SRC-1 (N-CoA1), GRIP1 (TIF2/N-CoA2), and ACTR (pCIP/AIB1/RAC3). These bind to the LBD only in the presence of cognate hormone, and their binding is blocked by antagonist ligands. The mechanism of binding is now understood in atomic detail and involves the docking of coactivator nuclear receptor boxes, which have the motif LXXLL with a hydrophobic cleft that forms on the surface of the hormone-bound LBD (2-6).The AF-1 domain of the estrogen, androgen, and perhaps other receptors also contacts the p160s but does so through surfaces outside of the nuclear receptor boxes (7,8).The p160s are complex proteins with multiple domains (Fig. 1). In addition to the nuclear receptor boxes they have two intrinsic transcriptional activation domains AD1 and AD2, whose activities may be monitored when the coactivators are directly tethered on DNA via fusion to a heterologous DNA binding domain (8, 9). AD1, which is essential for transcriptional mediation by p160s, is coextensive with the binding domain for the CBP/p300 family of coactivators. CBP/p300 complexes with the p160s and synergizes in coactivator function (10, 11). In particular, CBP/p300s contain a potent acetyltransferase activity that can transfer acetate from acetyl-CoA to histones and also to other proteins in the complex on DNA (12-18). AD2 contributes to coactivation by p160s in some circumstances and does so in part by binding CARM1 and other proteins that have histone methyltransferase activity (19). The coactivators are believed to mediate transcriptional activation by remodeling chromatin through their histone modification activities and also by direct effects on the transcriptional complex.In addition to regulating AF-2, hormones regulate steroid receptors (but not other nuclear r...