INITIAL INTRACELLULAR EVENTS OF STEROID HORMONE ACTION

Diabetes, Digestive, Kidney Diseases

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Abstract

A major focus of our research, and a crucial but poorly understood component of glucocorticoid hormone and steroid hormone action in general, is the determinants of the steroid hormone dose-response curve. Changes in the positioning of this curve, which defines the steroid concentrations required for the induction of target genes, can result in a differential control of gene expression. Our previous data established that a cis-acting element, called a GME, and its two associated novel binding proteins, GMEB-1 and -2, can shift the position of the dose-response curve for glucocorticoid receptor (GR) mediated transactivation of a gene under the control of a glucocorticoid response element (GRE). At the same time, the GME increased the residual agonist activity of antiglucocorticoids with the same GRE. These results shed new light on an additional conundrum of steroid hormone action: how the same receptor-antagonist complex can display different amounts of residual activity with different genes. In our continuing effort to understand the unusual activities of the GME, we have recently cloned the second of the two GME binding proteins as the 88 kDa human GMEB-1 and determined the genomic sequence of both the human GMEB-1 and the rat GMEB-2. The structure of the two genes, including portions of the introns, is highly conserved. However, GMEB-1 and -2 are found to reside on chromosomes 1 and 20 respectively, demonstrating that they are encoded by distinctly different genes. The tissue distribution of the each GMEB is not the same, with the levels of each being highest in fetal and developmental tissues. These results are consistent with previous suggestions that both homo- and heterooligomers may possess biological activities. This conclusion is supported by the finding that each protein, when fused to the GAL4 DNA binding domain, displays intrinsic transactivation activity with a GAL responsive reporter gene. GMEB-1 and -2 interact with themselves and each other in mammalian two hybrid and in pull-down assays. Overexpression of GMEB-1 and -2, either alone or in combination, results in a reversible right shift in the dose-response curve, and decreased agonist activity of antisteroids, as expected from the squelching of other limiting factors. We have recently described two other variables, in addition to the GME, that can reposition the dose-response curve of agonist-bound GRs and modify the quantity of partial agonist activity of antiglucocorticoids: GR concentration and coactivator concentration. Given these similar properties, we asked whether all three processes proceed via independent pathways or a common intermediate. Saturating levels of either GR or the coactivator TIF2 inhibit the ability of each protein, and the GME, to affect further changes in the dose-response curve or partial agonist activity of antisteroids. This competitive inhibition suggests that all three modulators proceed through a common step. Support for this hypothesis comes from the ability of both the viral protein E1A and a fragment of TIF2 to act as a dominant negative inhibitor of each variable (GME, GR, and coactivator). Collectively, these results suggest that three different inputs (GME, GR, and coactivator) for perturbing the dose-response curve, and partial agonist activity, of GR-steroid complexes act by converging at a single step that involves a limiting factor prior to transcription initiation. These combined findings contribute to our long term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.