Ligand binding domain mutations of the glucocorticoid receptor selectively modify the effects with, but not binding of, cofactors.

The ability of steroid receptors to regulate gene transcription lies at the heart of their roles in differential gene expression during development, differentiation, and homeostasis. Glucocorticoids are particularly effective because they affect virtually every cell in the body and are used to treat a variety of human pathologies including asthma, autoimmune diseases, cancer, and the induction of surfactant in the lungs of premature infants (1, 2). These effects of glucocorticoid steroids are thought to occur via the classical mechanism of passive diffusion into the cell and binding to the intracellular receptor protein. The resulting glucocorticoid receptor (GR1)-steroid complex binds with high affinity to biologically active DNA sequences to recruit other transcriptional cofactors, thereby increasing or decreasing the rates of transcription of gene expression. In addition to modifying the rates of transcription to increase or decrease the maximal levels of gene product (Amax), many transcriptional cofactors also alter both the concentration of agonist steroid required for half-maximal induction (EC50) and the partial agonist activity of antisteroids (PAA) (reviewed in 3, 4). The EC50 is a measure of steroid potency but it is not the same for all genes regulated by that steroid. Thus, the endogenous subsaturating amount of steroid will cause a different percentage of full induction (or repression) for most responsive genes. The PAA is expressed as percent of maximal activity of an agonist under the same conditions. Therefore, the PAA is a measure of the amount of residual agonist activity displayed by an antisteroid. This parameter is critical when antisteroids are used in endocrine therapies to block the action of endogenous agonist steroids. The PAA also varies with the gene examined. For this reason, it is theoretically possible during endocrine therapy to target the inhibitory action of antisteroids to a subset of the regulated genes, thereby reducing the number of unwanted side effects from that which occurs when all regulated genes are repressed. For all three parameters of GR-regulated gene expression (Amax, EC50, and PAA), changing the level of factor acts as a rheostat to give a continuum of responses for GRs and the other classical steroid receptors (3, 4). These two effects of changing cofactor concentration provide attractive additional mechanisms for achieving differential control of gene expression during development, differentiation, and homeostasis, i.e., by altering the responses in a gene-selective manner to subsaturating, physiological concentrations of agonist steroid and to pharmacologically administered doses of antisteroid. One frequently studied modulatory factor of GR transcriptional properties is TIF2/GRIP1. TIF2 (the name for the human coactivator) is a member of the p160 family of coactivators that includes SRC-1 and AIB1/pCIP/ACTR/TRAP1/RAC3. A central region of TIF2 containing three receptor interacting domains (RIDs) binds to a pocket in the AF2 activating function domain of the GR ligand binding domain (LBD), which is formed by helices 3, 4, 5, and 12 (5). More recently, a second, amino-terminal region of TIF2 has been found to interact with the amino-terminal domain of GR and inhibit corepressor binding to GR (6). Even with two GR-interaction domains for TIF2, there appears to be a limit to how much the Amax, EC50, and PAA of GR-mediated transactivation can be altered with increasing amounts of TIF2 (7). Of further interest is the recent report that several GR LBD mutations in the steroid binding pocket (8) selectively alter both the absolute Amax and EC50 of GR-mediated gene induction in a steroid-specific manner (9). These GR mutations were chosen following a comparison of the x-ray structures of GR LBD bound by two very differently shaped steroids: dexamethasone (Dex) and deacylcortivazol (DAC) (8). It was predicted that these mutations would selectively affect Dex vs. DAC binding. Unexpectedly, the changes in steroid binding of many mutants were minor compared to their effects on Amax, EC50, and PAA (9). This was proposed to result in part from alterations in specific GR surfaces. These perturbed surfaces did not affect TIF2 binding to GR but did modify, via protein-protein induced changes, some TIF2 domains interacting with downstream transcription factors (9). This observation led us to ask if such changes in the efficiency of information transmission through a GR-bound cofactor to the transcriptional machinery was unique to TIF2 or also occurred with other factors known to influence GR transactivation properties. The phenomenon would be of even greater interest if it also occurred for a cofactor that bound to a different region of GR (10). Such a result would suggest that subtle changes in LBD structure can have wider spread topological consequences (11, 12) and are not limited to the GR-TIF2 contact surface(s). A particularly attractive cofactor with which to address this question is Ubc9, which is the human homolog of a yeast E2-ubiquitin conjugating enzyme. Ubc9 mediates the attachment of a small ubiquitin-like molecule (SUMO) to proteins. However, Ubc9 also alters the Amax, EC50, and PAA of GR-regulated transcription in a manner that is independent of its sumoylation activity (13–15). Like TIF2, Ubc9 binds to the GR LBD (10, 14). Like TIF2, Ubc9 action depends upon domains in both the N-terminal half and LBD of GR, although the LBD interactions are the strongest (14). However, there are two important differences between Ubc9 and TIF2. First, the modulatory effects of TIF2 are most dramatic at low, and disappear at high, GR concentrations (7). In contrast, Ubc9 increases the Amax at low and high concentrations of GR but reduces the EC50, and enhances the PAA, only with high GR concentrations (13–16). Second, the effects of Ubc9 appear to be manifested at a step downstream of TIF2 action (13, 16). This argues that TIF2 and Ubc9 do not interact with the same factors to modulate Amax, EC50, or PAA and could contact different surfaces of the GR LBD. The purpose of this study was to determine whether the above receptor mutations that alter GR activity with TIF2 also affect the activity of two other cofactors that are known to modulate the transactivation properties of GR but bind to different regions of the GR LBD, and/or interact with other cofactors, than TIF2. The first factor is Ubc9. The second is GR itself. It is well documented that increasing concentrations of GR elevate the PAA and decrease the EC50 of GR gene transactivation (3, 7, 17). GR also forms dimers using surfaces removed from that for binding TIF2 (18). We now find that, when the concentration of GR or Ubc9 is increased, there are minimal consequences of these LBD mutations on Amax but much larger reductions in PAA and EC50. In some circumstances, changes in EC50 depend upon the structure of the bound agonist steroid. These results, in combination with our previous data with TIF2, lead us to conclude that minor changes in GR LBD structure can influence the activity, but not the binding, of a variety of associated transcriptional cofactors in a manner that is sometimes further influenced by the nature of the agonist steroid.