The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERalpha (hERalpha) is enhanced through phosphorylation of the Ser(118) residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERalpha. Phosphorylation of hERalpha Ser(118) potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERalpha in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERbeta, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERalpha AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERalpha A/B domain was essential for the full activity of hERalpha AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERalpha AF-1 and strongly suggest that the interaction between p68 and the hERalpha A/B domain is regulated by MAPK-induced phosphorylation of Ser(118).

Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-beta) and the liphophilic hormone vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways.

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identi®ed a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor a (hERa) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hERa A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogenbound hERa in MCF7 cells and in partially puri®ed complexes associated with hERa from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hERa and TIF2 in the nucleus. The presence of p72/ p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hERa. These ®nd-ings indicate that p72/p68 acts as an ER subtypeselective coactivator through ERa AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.

Recent studies have shown that adiponectin, an adipocyte-derived cytokine, acts as a potent inhibitor of inflammatory responses. It has been also demonstrated that bacterial and viral signalings in host cells are triggered via Toll-like receptor (TLR) molecules. Therefore, in the present study, we investigated whether globular adiponectin (gAd) would be able to inhibit TLR-mediated nuclear factor-jB (NF-jB) signaling in mouse macrophages (RAW264). gAd predominantly bound to the AdipoR1 receptor and suppressed TLR-mediated NF-jB signaling. gAd-mediated inhibition of TLR-mediated IjB phosphorylation and NF-jB activation was eliminated by the pretreatment of cycloheximide. Also their inhibitions of gAd were blocked by preincubation of the cells with an antibody against AdipoR1, but not with an antibody against AdipoR2. Taken together, these findings indicate that adiponectin negatively regulates macrophage-like cell response to TLR ligands via an unknown endogenous product(s).

Estrogen receptor (ER)␣

For the transactivation function of VDR, only the ligand-binding domain (E region) is thought to be responsible in a ligandbinding-dependent way (27), although two transactivation domains, one at the N terminus (AF-1) and one at the C terminus (AF-2), are present in most nuclear receptors. To achieve ligand-induced transactivation, the nuclear receptors recruit several nuclear receptor coactivators. They include members of the SRC-1/TIF2 family (38, 48), CBP/p300 (9, 29), and RIP140 (8). Members of the SRC-1/TIF2 family [SRC-1 (p160, ERAP160) (18, 23), TIF2 (Grip-1) (10), and AIB-1 (ACTR) (3)] mediate the function of the AF-2 of the nuclear receptors, and the interaction site has been mapped to the minimal activation domain (AD) of AF-2 (13, 23, 50). Interestingly, it was recently shown that the interactions of estrogen receptor with SRC-1 or TIF2 are induced by estrogen (E 2 ) but not by its antagonists, tamoxifen and ICI164,384. These findings indicate that the structure of the ligand-bound E region recruiting coactivators is ligand specific (18). This idea is further supported by recent findings from crystallographic analysis that the position of the AF-2 AD (helix 12) in the estrogen receptor E region which binds the E 2 antagonists clearly differs from the one which binds E 2 (17). From the structural similarity of the ligand-binding domains of nuclear receptors, ligand type-specific alterations in their structures, at least in the helix 12 positions, are postulated (7).Several synthetic 1␣,25(OH) 2 D 3 derivatives, such as F 6 -1␣,25-(OH) 2 D 3 [26,26,26,27,27,25(OH) OCT (25,40,44). Taking these facts together, it is reasonable to speculate that the selective interactions of VDR with coactivators induced by vitamin D analogs specify the biological activities of the vitamin D analogs. Such differential combinations of transcription factors and coactivators are believed to activate only particular sets of target gene promoters (46).To test this possibility, we studied the interaction of vitamin D analog-bound VDR with nuclear receptor coactivators and interacting factors. We found that although the in vivo and in vitro interactions of VDR with SRC-1, TIF2, and AIB-1 were induced by F 6 -1␣,25(OH) 2 D 3 and ED-71 as well as by 1␣,25 (OH) 2 D 3 , OCT induced interaction only with TIF2. Such interactions were also observed in the VDR-RXR heterodimer bound to DNA. Consistent with the interactions, only TIF2 potentiated the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the VDR structure is altered in a vitamin D analog-specific way, resulting in selective interaction of VDR with coactivators. Such selective coactivator interaction with VDR may specify the array of biological actions of a vitamin D analog such as OCT, possibly through activating a particular set of target gene promoters. MATERIALS AND METHODSYeast two-hybrid system and ␤-galactosidase assay. The pGBT9(GAL4-DBD)-VDR(DEF) fusion plasmid was constructed by inserting rat VDR-DEF regions (encoding amino acids ...

The nuclear peroxisome proliferator-activated receptor ␥ (PPAR␥) is a member of the nuclear receptor superfamily and acts as a ligand-dependent transcription factor mediating adipocyte differentiation, cell proliferation and inflammatory processes, and modulation of insulin sensitivity. Members of the 160-kDa protein (SRC-1/TIF2/AIB-1) family of coactivators, CBP/p300 and TRAP220/DRIP205, are shown to interact directly with PPAR␥ and potentiate nuclear receptor transactivation function in a ligand-dependent fashion. Because PPAR␥ ligands exert partially overlapping but distinct subsets of biological action through PPAR␥ binding, we wished to examine whether interactions between PPAR␥ and known coactivators were induced to the same extent by different classes of PPAR␥ ligand. The natural ligand 15-deoxy-⌬12,14-prostaglandin J 2 induced PPAR␥ interactions with all coactivators tested (SRC-1, TIF2, AIB-1, p300, TRAP220/DRIP205) in yeast and mammalian twohybrid assays, as well as in a glutathione S-transferase pull-down assay. However, under the same conditions troglitazone, a synthetic PPAR␥ ligand that acts as an antidiabetic agent, did not induce PPAR␥ interactions with any of the coactivators. Our findings suggest that ligand binding may alter PPAR␥ structure in a ligand type-specific way, resulting in distinct PPAR␥-coactivator interactions.Peroxisome proliferator-activated receptor ␥ (PPAR␥), 1 a member of the nuclear hormone receptor superfamily, acts as a ligand-inducible transcription factor (1, 2). PPAR␥ forms a heterodimer complex with one of the three retinoid X receptor (RXR) proteins, which then binds to PPAR-responsive elements (PPRE) within the promoters of PPAR␥ target genes (3, 4). It is thought that the ligand binding domain (LBD) mediates the ligand-dependent transactivation function of PPAR␥, although two transactivation domains, at the N-terminal (AF-1) and C-terminal ends (AF-2), are present in most nuclear receptors. Ligand-induced transactivation is achieved by the nuclear receptor recruiting one of several types of nuclear receptor coactivator complex. One class of coactivator complex includes three SRC-1 family members (5), CBP/p300 (6), and SRA (7), as well as other proteins (8, 9). The SRC-1 family members (SRC-1 (p160/NCoA-1) (10), TIF2 (GRIP-2) (11), and AIB1 (p/CIP/ ACTR) (12)) interact with the AF-2 nuclear receptors. This interaction is highly ligand-dependent through direct binding to the minimal activation domain of AF-2 (AF-2 AD), mapped to the C-terminal ␣-helix 12 (H12) in the LBD (13). CBP/p300 serves as an essential coactivator not only for nuclear receptors but also for other classes of transcription regulatory factor (14) and, like the SRC-1 family members, possesses histone acetyltransferase activity (15). Another coactivator complex, TRAP/ DRIP, contains at least 12 components, one of which exhibits direct and ligand-dependent interaction with H12 in the LBD (TRAP220/DRIP205/PBP) (16,17). Reflecting on the role of the PPAR␥ function in many biological events, a variety of...