Upon binding of ligand and translocation to the nucleus, SHRs bind to specific regions in the DNA called Hormone Responsive Elements (HREs) through zinc-finger motifs present in the DBD (, step 5). The exact mode of binding has been characterised in detail with help of available crystal structures and extensive biophysical in vitro measurements. Consensus nucleotide binding sequences have been determined for all SHRs, but these show a significant amount of ambiguity, making it hard to pinpoint true target HREs in the genome. A HRE is made up of two so-called half-sites that each bind one monomer of the SHR dimer. Interestingly, single half-sites have also been found in genes that clearly respond to hormone, hinting at a possible role for receptors in their monomeric configuration.
Immobilization of SHRs on DNA and other nuclear structures has been studied with photobleaching techniques like Fluorescence Recovery After Photobleaching (FRAP). By bleaching fluorescent molecules in a region of interest in a living cell and measuring recurrence of fluorescence levels in the bleached area, the mobility of the tagged molecules can be determined (). Using this technique several groups were able to demonstrate a clear correlation between receptor immobilization in the nucleus and the appearance of the typical punctate receptor distribution, which was most convincingly demonstrated by Schaaf and colleagues who compared 13 GR ligands [Schaaf et al., 2005
]. FRAP measurements show that fluorescently tagged SHRs such as ER, GR, and AR are highly mobile and dynamic in the unliganded state, whereas ligand-bound forms are less mobile [Farla et al., 2004
; Sprague et al., 2004
; Stenoien et al., 2001b
]. Stenoien et al., further showed that in the case of ERα, FRAP could discriminate between ligands with potential agonistic properties and full antagonists on the basis of receptor immobilization in the nucleus [Stenoien et al., 2001b
]. The nature of the substrate on which the receptor immobilizes remains uncertain, but almost certainly includes DNA. Carefully controlled FRAP measurements from Sprague et al., show that in free form GR is bound to a single type of substrate, most probably DNA, with each molecule binding on average 65 sites per second [Sprague et al., 2004
]. This rapid sampling of GR is likely to be important in finding a specific HRE. Upon ligand binding, the residence time on DNA is significantly increased. According to Farla et al., on average one out of five ARs is immobilized in the presence of ligand, each individual AR being immobile for 1-2 min. This immobilization is dependent on DNA binding since GFP-ARs mutated in the DNA-binding domain were not immobilized [Farla et al., 2004
]. Likewise, FRAP analysis by Kino et al., on several GR mutant receptors showed a significantly increased nuclear motility and decreased chromatin retention, which correlated with impaired transcriptional activity [Kino et al., 2004
Fluorescence recovery after photobleaching (FRAP).
DNA binding and transcription has been visualized directly by using cells that have stably integrated a tandem array of HREs. Pioneering work in this area has been performed by the Hager lab, which used this approach to study the interaction of GR with a natural promoter [McNally et al., 2000
]. The promoter array allows significant amounts of GFP-GR to accumulate for direct detection under the microscope. The recruitment of GFP-GR leads to gross alterations in chromatin structure of the array that correlate with gene transcription [Muller et al., 2001
]. Interestingly, FRAP analysis on the array again shows a rapid exchange of receptors between chromatin and the nucleoplasmic compartment. Further analysis demonstrated that following binding of GR to the promoter, the receptor is actively displaced from the template during a chromatin remodeling reaction facilitated by the hSWI/SNF complex [Nagaich et al., 2004
]. Further evidence comes from work on PR by the same group, which showed that the exchange of PR-GFP on the array was slowed down (but still in the order of seconds) upon agonist addition, and even further slowed down after addition of a partial antagonist [Rayasam et al., 2005
]. Strikingly, addition of a full-antagonist showed the opposite effect, with ongoing exchange at a rate faster than for an agonist bound receptor. In contrast to an agonist or partial antagonist bound receptor, addition of a full-antagonist does not lead to recruitment of the SWI/SNF chromatin remodeling complex, which may partly explain the above results. Together, these findings have led to the so-called hit-and-run model. In contrast to static binding of the receptor to a HRE and the subsequent build up of the transcription complex, this model suggests a receptor continuously probes the DNA for potential binding sites. Transcriptional activation reflects the probability that all components required for activation will meet at a certain chromatin site.
Besides binding to Hormone Responsive Elements, SHRs can also exert their effects by binding directly to other transcription factors. For example, ERα is able to bind to fos/jun, and thus regulate AP-1 mediated transcription of genes like cyclin D1. Similarly, ERα can bind Sp1 proteins and regulate transcription of genes that contain a Sp1/ER binding site. Interestingly, antagonists often have agonistic effects in this setting, which may be important when it comes to resistance to antagonistic compounds. This is illustrated by work from Kim et al., who used FRET to visualize the interaction between ERα and Sp1 [Kim et al., 2005
]. Addition of the full anti-estrogen ICI 182,780 inhibits normal ERα mediated transcription, yet like agonist estradiol induced a FRET signal between ERα and Sp1 that correlated with Sp1 mediated transcription of a reporter construct.
Recently a number of groups have claimed a role for SHRs in non-genomic, extranuclear signalling events (, step 6). Targeting ERα artificially to the plasma membrane has a marked influence on ERK1/2 signalling, which was not affected by full anti-estrogens [Rai et al., 2005
]. Similar effects on the Mitogen Activated Protein Kinase (MAPK) and Protein Kinase A (PKA) pathways have also been attributed to the wildtype receptor [Levin, 2005
; Razandi et al., 2004
]. However, most studies are based on biochemical approaches where post-lysis artefacts are hard to exclude. Moreover, convincing microscopic pictures of SHR membrane localization are still lacking. Nevertheless, accumulating evidence seems to point to possible functions for SHRs other than those mediated by DNA binding.