Unlinked Regulation of the Sensitivity of Primary Glucocorticoid-Inducible Responses in Mouse Mammary Tumor Virus Infected Fu5-5 Rat Hepatoma Cells

Self Cite

The underlying molecular mechanism for the expression of agonist versus antagonist activity for a given receptor-steroid complex is still not known. One attractive hypothesis, based on data from progesterone receptors, is that agonist versus antagonist binding induces unique conformations at the C terminus of receptors, which can be detected by the different fragments produced by partial proteolysis. We now report that the determinants of glucocorticoid receptor (GR)-antagonist complex activity are more complex. Steroid binding did cause a conformational change in the GR that was detected by partial trypsin digestion, as described previously (Simons, S. S., Jr., Sistare, F. D., and Chakraborti, P. K. (1989) J. Biol. Chem. 264, 14493-14497). However, there was no uniformity in the digestion patterns of unactivated or activated receptors bound by a series of six structurally different antagonists including the affinity labeling antiglucocorticoid dexamethasone 21-mesylate. A total of four resistant bands were observed on SDS-polyacrylamide gels in the range of 30 -27 kDa. Using a series of point mutations and epitope-specific antibodies, it was determined that the 30-kDa species represented the entire C-terminal sequence of amino acids 518 -795, whereas the other bands arose from additional N-terminal and/or C-terminal cleavages. Bioassays with GRs containing various point and deletion mutations failed to reveal any Cterminal alterations that could convert antagonists into biologically active agonists. Thus, the presence or absence of C-terminal amino acids of the GR did not uniquely determine either the appearance of smaller trypsin-resistant fragments or the nature of the biological response of receptor-bound antisteroids. When compared with the current model of the ligand-binding domain, which is based on the x-ray structures of the comparable region of thyroid and retinoic acid receptors, the present results suggest that sequences outside of the model structure are relevant for the binding and biological activity of GRs.

Section: Discussionmentioning

confidence: 99%

Steroid-induced Conformational Changes at Ends of the Hormone-binding Domain in the Rat Glucocorticoid Receptor Are Independent of Agonist Versus Antagonist Activity

Modarress¹,

Opoku²,

et al. 1997

Self Cite

“…The EC 50 was initially thought to be determined by the affinity of steroid binding to cognate receptor (18 -20). However, reports of different dose-response curves for various genes within a cell for the same receptor-steroid complex indicate the involvement of additional parameters (21)(22)(23)(24)(25)(26)(27). Furthermore, the EC 50 can even change for the same gene under different cellular conditions (28,29).…”

mentioning

confidence: 99%

Structure/Activity Elements of the Multifunctional Protein, GMEB-1

Kaul

Simons

2002

Self Cite

GMEB-1 was initially described as a component of a 550-kDa heteromeric DNA binding complex that is involved in the modulation of two properties of glucocorticoid receptor (GR) transactivation, the dose-response curve of agonists and the partial agonist activity of antagonists. Subsequently, GMEB-1 was also found to bind to hsp27, to associate with the coactivator TIF2 in yeast cells, and to participate in Parvovirus replication. To understand these multiple activities of GMEB-1 at a molecular level, we have now determined which regions are associated with the various activities associated with the modulation of GR transactivation properties. These activities include, homooligomerization, heterooligomerization, DNA binding, binding to GR and the transcriptional cofactor CBP, and GR modulation. Complex activities such as DNA binding and GR modulation, are found to require the physical combination of those domains that would be predicted from the involved biochemical processes. We have previously documented that GMEB-1 possesses both GR modulatory and intrinsic transactivation activity. However, the domains for these two activities of GMEB-1 are found not to overlap. This separation of activities provides a structural basis for our prior biological observations that the modulation of the dose-response curve and partial agonist activity of GR complexes is independent of the total levels of gene activation by the same GR complexes.

“…Whereas those mechanisms regulating the level of response to saturating concentrations of agonist steroids should persist at subsaturating concentrations, thereby leaving the EC 50 of the dose-response curve unaffected, the converse is not necessarily true. In fact, we have reported that the doseresponse curve for TAT gene induction in Fu5-5 cells is leftshifted (to give a lower EC 50 ) relative to the same gene in HTC cells (18 -21) or to a different gene in the same Fu5-5 cells (19,(21)(22)(23)(24). Thus, physiological concentrations of glucocorticoid elicited a greater percentage of the maximal induction of the TAT gene in Fu5-5 cells than of any other genes examined.…”

mentioning

confidence: 99%

Cloning and Characterization of a Novel Binding Factor (GMEB-2) of the Glucocorticoid Modulatory Element

Zeng

Jackson²,

Oshima³

et al. 1998

Self Cite

The 21-base pair glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is the only cis-acting element known to modulate the transcriptional activity of receptors bound to glucocorticoid response elements. Specifically, the GME increases the activity of complexes bound both by physiological concentrations of glucocorticoids, due to a left shift in the dose-response curve, and by saturating concentrations of anti-glucocorticoids. For this reason, the nuclear protein(s) that has been demonstrated to bind to the GME is of major interest as a possible transcription factor with hitherto undescribed properties. Subsequent studies indicated that not one but two proteins of 88 and 67 kDa ‫؍(‬ GMEB-1 and -2, respectively) formed a heteromeric complex with double-stranded GME oligonucleotides in gel shift assays and participated in the expression of GME activity (Oshima, H., Szapary, D., and Simons, S. S., Jr. (1995) J. Biol. Chem. 270, 21893-21910). Here, we report the use of polymerase chain reaction of degenerate oligonucleotides and 5-and 3-rapid amplification of cDNA ends to clone two cDNAs of 2.0 and 1.9 kilobase pairs that probably result from alternative splicing. Both cDNAs encoded open reading frames containing all four previously sequenced peptides. The longer 2.0-kilobase pair cDNA encoded an open reading frame for an acidic, 529-amino acid protein and afforded a major 67-kDa and a minor 58-kDa protein after in vitro transcription/translation. Both proteins were recognized by a mono-epitopic antibody raised against a peptide of GMEB-2. The in vitro translated protein bound to GME DNA in gel shift assays. However, the binding to GME DNA increased markedly after mixing with authentic GMEB-1 to give a gel-shifted complex that was similar to that derived from HTC cell cytosol. GMEB-2 shares a unique domain (KDWKR) with proteins derived from diverse organisms as follows: Drosophila (DEAF-I), rat (Suppressin), and Caenorhabditis elegans (three unknown open reading frames). Collectively, these data suggest that the 67-kDa GMEB-2 not only is an important factor for the modulation of glucocorticoid receptor bound to glucocorticoid response elements but also may belong to a novel family of transcription factors.