No abstract

A distinguishing, but unexplained, characteristic of steroid hormone action is the dose-response curve for the regulation of gene expression. We have previously reported that the dose-response curve for glucocorticoid induction of a transfected reporter gene in CV-1 and HeLa cells is repositioned in the presence of increasing concentrations of glucocorticoid receptors (GRs). This behavior is now shown to be independent of the reporter, promoter, or enhancer, consistent with our proposal that a transacting factor(s) was being titrated by added receptors. Candidate factors have been identified by the observation that changes in glucocorticoid induction parameters in CV-1 cells could be reproduced by varying the cellular levels of coactivators [transcriptional intermediary factor 2 (TIF2), steroid receptor coactivator 1 (SRC-1), and amplified in breast cancer 1 (AIB1)], comodulator [CREB-binding protein (CBP)], or corepressor [silencing mediator for retinoid and thyroid-hormone receptors (SMRT)] without concomitant increases in GR. Significantly, the effects of TIF2 and SMRT were mutually antagonistic. Similarly, additional SMRT could reverse the action of increased levels of GRs in HeLa cells, thus indicating that the effects of cofactors on transcription may be general for GR in a variety of cells. These data further indicate that GRs are yet an additional target of the corepressor SMRT. At the same time, these cofactors were found to be capable of regulating the level of residual agonist activity displayed by antiglucocorticoids. Finally, these observations suggest that a novel role for cofactors is to participate in processes that determine the dose-response curve, and partial agonist activity, of GR-steroid complexes. This new activity of cofactors is disconnected from their ability to increase or decrease GR transactivation. An equilibrium model is proposed in which the ratio of coactivator-corepressor bound to either receptor-agonist or -antagonist complexes regulates the final transcriptional properties.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a rgceptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high-and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.The protein tyrosine kinase gene family includes a group of proteins which act as membrane receptors for growth factors (for a review, see reference 6). The enzymatic activity of the cytoplasmic kinase domain of these receptors is regulated by the binding of specific growth factors to the extracellular-ligand-binding domain (for a review, see references 6 and 13). This leads to receptor self-phosphorylation and to the phosphorylation of specific substrates which may play a role in the pleiotropic response leading to mitogenesis. All protein tyrosine kinases contain a consensus lysine residue in the kinase domain which is thought to function as part of the ATP-binding site (3,4,16,17) (i.e., in epidermal growth factor receptor [EGF-R] or Lys-1018 in insulin receptor). It has been shown that mutations at these consensus lysine residues abolish the protein tyrosine kinase activity of insulin receptor (4) and of other members of the src gene family (7,15,19). To examine the role of the kinase activity of EGF-R, we have prepared an Ala-721 EGF-R cDNA construct by in vitro site-directed mutagenesis (5). The Ala-721 EGF-R cDNA construct was cloned into a mammalian expression vector which utilizes the simian virus 40 early promoter for gene expression and also contains the dihydrofolate reductase and neomycin resistance genes as selectable markers (12). The plasmid was transfected into NIH-3T3 cells, and Geneticin G418 (GIBCO) was used for selection of cells expressing receptor molecules. Several cloned cell lines expressing the wild-type receptor on the Ala-721 receptor mutant were developed. The NIH-3T3 cells used for transfection were obtained from C. Fryling. We compared various clones of NIH-3T3 cells for the expression of EGF-R by using immunoprecipitation and 125I-labeled

Transient transfections of steroid receptors have yielded much of the data used to construct the current models of steroid hormone action. These experiments invariably examine the ability of receptors to regulate transcription when occupied by saturating concentrations of steroid. We now report that other induction properties of a transiently transfected gene are not constant but vary with the concentration of transiently transfected glucocorticoid receptors. Thus, the percentage of maximal induction seen with subsaturating concentrations of glucocorticoid could be dramatically increased, and an antiglucocorticoid could be converted into a partial glucocorticoid, simply by increasing the concentration of glucocorticoid receptors. This behavior was observed in HeLa cells, containing endogenous receptors, or in CV-1 cells, containing almost no endogenous receptor, with either homologous or heterologous receptors. These increases were relatively insensitive to the concentration of reporter gene, suggesting the titration of some transcription factor(s) involved in regulating the position of the glucocorticoid dose-response curve and the agonist activity of an antiglucocorticoid. This property of transfected glucocorticoid receptors required a full-length, functionally active receptor but was retained, albeit reduced in magnitude, in the absence of binding to a glucocorticoid response element. Furthermore, this phenomenon was specific in that the A form of the human progesterone receptor had no effect under the same conditions. These variations in induction properties of antiglucocorticoids and of subsaturating concentrations of glucocorticoid, in a manner that was proportional to the amount of transfected receptor, reveal processes that are not operative with saturating concentrations of glucocorticoid. These variations also demonstrate that caution should be exercised in making mechanistic conclusions based solely on experiments conducted with saturating concentrations of glucocorticoid.The overriding experimental advantage of transient transfections is time. Thus, the biological consequences of altered nucleotide compositions in the cDNAs encoding active proteins, and in genomic sequences, can be examined in a fraction of the time required to establish cell lines with the same sequences stably integrated into the cellular genome. In the field of steroid receptors, most of the recent advances have emerged from transient transfection experiments, including the contributions of cis-acting elements (1, 2), of different nucleotides in receptor binding to the hormone-responsive element (3), of various regions of receptors in steroid binding and biological activity (reviewed in Ref. 4), of promoter structure and cell type as determinants for the activity of antisteroid activity (5), and of overlapping signaling systems such as dopamine (6), epidermal growth factor (7), and protein kinase A inducers (8, 9). The utility of transient transfections has been further enhanced by the development of the "two-hybrid" (10, 11) and...

In vitro site-directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF) -

Epidermal growth factor (EGF)‐receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH‐3T3 cells lacking endogenous EGF‐receptors. Kinetic parameters of the kinase of wild‐type and mutant receptors were compared. Both wild‐type and mutant EGF‐receptors had a Km(ATP) 1‐3 microM for the autophosphorylation reaction, and a Km(ATP) of 3‐7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF‐receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF‐receptor (KGSTAENAEYLRV) was phosphorylated by wild‐type receptor with a Km of 110‐130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF‐receptors phosphorylated the peptide substrate with a Km of 70‐100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c‐src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild‐type receptor to 890 microM for mutant receptor and for c‐src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650‐750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Coactivators such as TIF2 and SRC-1 modulate the positioning of the dose-response curve for agonistbound glucocorticoid receptors (GRs) and the partial agonist activity of antiglucocorticoid complexes. These properties of coactivators differ from their initially defined activities of binding to, and increasing the total levels of transactivation by, agonist-bound steroid receptors. We now report that constructs of TIF2 and SRC-1 lacking the two activation domains (AD1 and AD2) have significantly less ability to increase transactivation but retain most of the activity for modulating the dose-response curve and partial agonist activity. Mammalian two-hybrid experiments show that the minimum TIF2 segment with modulatory activity (TIF2.4) does not interact with p300, CREB-binding protein, or PCAF, which also modulates GR activities. DRIP150 and DRIP205 have been implicated in coactivator actions but are unable to modulate GR activities. The absence of synergism by PCAF or DRIP150 with SRC-1 or TIF2, respectively, further suggests that these other factors are not involved. The ability of a TIF2.4 fragment (i.e. TIF2.37), which is not known to interact with proteins, to block the actions of TIF2.4 suggests that an unidentified binder mediates the modulatory activity of TIF2. Pull-down experiments with GST/TIF2.4 demonstrate a direct interaction of TIF2 with GR in a hormone-dependent fashion that requires the receptor interaction domains of TIF2 and is equally robust with agonists and most antiglucocorticoids. These observations, which are confirmed in mammalian two-hybrid assays, suggest that the capacity of coactivators such as TIF2 to modulate the partial agonist activity of antisteroids is mediated by the binding of coactivators to GR-antagonist complexes. In conclusion, the modulatory activity of coactivators with GR-agonist and -antagonist complexes is mechanistically distinct from the ability of coactivators to augment the total levels of transactivation and appears to involve the binding to both GR-steroid complexes and an unidentified TIF2-associated factor(s).

Glucocorticoid induction of the tyrosine aminotransferase gene deviates from that of many glucocorticoidresponsive genes by having a lower EC 50 and displaying more agonist activity with a given antiglucocorticoid. A cis-acting element, located 3646 base pairs upstream of the start of tyrosine aminotransferase gene transcription, has been found to be sufficient to reproduce these variations with heterologous genes and promoters (Oshima, H., and Simons, S. S., Jr. (1992) Mol. Endocrinol. 6, 416 -428). This element has been called a glucocorticoid modulatory element, or GME. Others have called this sequence a cyclic AMP-responsive element (CRE) due to the binding of the cyclic AMP response element binding protein (CREB). We now report the partial purification and characterization of two new proteins (GMEB1 and -2) of 88 and 67 kDa that bind to the GME/ CRE as a heteromeric complex. This purification was followed by the formation of a previously characterized, biologically relevant band in gel shift assays. By several biochemical criteria, the GMEBs differed from many of the previously described CREB/CREM/ATF family members. Partial peptide sequencing revealed that the sequences of these two proteins have not yet been described. Size exclusion chromatography and molecular weight measurements of the gel-shifted band demonstrated that the GMEBs bound to the GME as a macromolecular complex of about 550 kDa that could be dissociated by deoxycholate. Similar experiments showed that CREB bound to the GME as heteromeric complexes of about 310 and 360 kDa. As determined from gel shift assays, GMEB1 and -2 are not restricted to rat liver cells but appear to be ubiquitous. Thus, these novel GMEBs may participate in a similar modulation of other glucocorticoid-inducible genes in a variety of cells.