The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) Steroid hormone receptors belong to a structurally and functionally related group of intracellular proteins, known as the nuclear receptor or steroid/thyroid hormone receptor superfamily, that serve as ligand-activated transcriptional regulators (1). Binding of the cognate hormone to steroid receptors causes a conformational change that allows the receptors to dissociate from an inhibitory complex of proteins, bind as dimers to specific regulatory sequences (enhancer elements) that are associated with the target genes regulated by the hormone, and modulate the transcription of the target genes. DNA binding by hormone-activated steroid receptors has been shown to cause chromatin remodeling, but the mechanism of transcriptional regulation is also believed to involve some type of direct or indirect interaction of the DNA-bound receptor with the transcription machinery.Like other nuclear receptors, steroid receptors are composed of three major functional domains: an N-terminal transcriptional activation domain (AD), a central DNA binding domain (DBD), and a C-terminal hormone binding domain (HBD) (1, 2). In spite of this nomenclature, both the Nterminal AD and the HBD contribute to the transcriptional activation function of steroid receptors (3, 4). In the absence of the HBD, the N-terminal AD, called AF-1, can function in a hormone-independent manner in mammalian cells. In contrast, the transcriptional activation function of the HBD, called AF-2, is hormone dependent. Although each isolated AD has some activity in mammalian cells, these two together appear to function synergistically.The mechanism by which DNA-bound steroid receptors can activate transcription initiation from associated promoters is still unknown. It is proposed that DNA-binding transcriptional activator proteins, including the steroid receptors, stimulate the efficiency of transcription initiation by RNA polymerase II by either directly or indirectly affecting the assembly of basal transcription factors into a preinitiation complex (1, 5). In addition to RNA polymerase II, the preinitiation complex consists of seven basal transcription factors, namely TFIIA, TFIIB, TATA-box binding protein (a subunit of TFIID), TFIIE, TFIIF, TFIIH, and TFIIJ. This complex alone can initiate transcription at a basal rate from TATA-containing promoters, whereas additional TFIID subunits are required for TATA-less promoters and for enhancer-activated transcription. Some of the basal transcription factors may be the targets for regulation by DNA-binding transcriptional activator proteins. Although examples of direct interaction between basal transcription factors and some DNA-binding transcriptional activators have been reported, in most cases the DNAbound transcriptional activator proteins require ...

After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor ␣, vitamin D receptor, retinoic acid receptor ␣, and retinoid X receptor ␣. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.Nuclear hormone receptors (NRs) are conditional transcription factors that play important roles in various aspects of cell growth, development, and homeostasis by controlling expression of specific genes (4,15,29,43). Members of the NR superfamily, which includes the five steroid receptors (SRs) as well as the receptors for thyroid hormone (TR), retinoic acid (RAR), and vitamin D (VDR), are structurally characterized by three distinct domains: an N-terminal transcriptional activation domain (AD), a central DNA binding domain (DBD), and a C-terminal hormone binding domain (HBD). Before the binding of hormone, SRs, which are sometimes called class I of the NR family, remain inactive in a complex with hsp90 and other stress family proteins. The binding of hormone induces critical conformational changes in SRs that cause them to dissociate from the inhibitory complex, bind as homodimers to specific DNA enhancer elements associated with target genes, and m...

The tau 2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding tau 2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the tau 2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the tau 2-region of the mouse glucocorticoid receptor (mGR, amino acids 533-562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum tau 2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum tau 2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum tau 2-fragment and the full-length GR indicated that the tau 2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the tau 2-region (mGR 541-547) was primarily involved in hormone binding, whereas the C-terminal portion of the tau 2-region (mGR 548-561) was primarily involved in transactivation.

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The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.