Steroid hormone receptor - an overview | ScienceDirect Topics
Transcription controlled by Steroid Hormone Receptors SHRs plays a key role in many important physiological processes like organ development, metabolite homeostasis, and response to external stimuli. Understandably, the members of this family have drawn a lot of attention from the scientific community since their discovery, four decades ago.
Still, after many years of research we are only beginning to unravel the complex nature of these receptors. The pace at which we do has improved significantly in recent years with the discovery of genetically encoded fluorescent probes, and the accompanying revival of biophysical approaches that allow more detailed study of SHRs. Here, we will look into the different aspects of SHR signalling, and discuss how biophysical techniques have contributed to visualizing their function in their native context, the living cell.
As early as , Thomas Beatson described that removal of the ovaries in advanced breast cancer patients often resulted in remarkable improvement [ Beatson, ].
With that he had revealed the stimulating effect of the female ovarian hormone estrogen on breast cancer, even before the hormone itself was discovered.
His work provided a foundation for the modern use of hormone therapy in treatment and prevention of breast cancer. Only much later was the cellular counterpart that mediated the described effects revealed, the estrogen receptor ER [ Green et al.
As it turned out, this receptor plays a key role in the development and maintenance of the sexual reproductive tissues, and therefore, as Beatson had discovered, in breast cancer as well. Phylogenetic tree of the Steroid Hormone Receptor SHR family showing the evolutionary interrelationships and distance between the various receptors. Numbers represent the length of the receptor in amino acids. All SHRs function as nuclear transcription factors whose activity is regulated by small lipid-soluble ligands, and each member plays an important role in key physiological processes like reproduction, glucose metabolism, salt balance, and stress response.
The members of the Steroid Hormone Receptor family share a similar, modular architecture, consisting of a number of independent functional domains Figure 1 B. Besides its ligand binding capability, the LBD also plays an important role in nuclear translocation, chaperone binding, receptor dimerization, and coregulator recruitment through its potent ligand-dependent transactivation domain, referred to as AF A second, ligand independent, transactivation domain is located in the more variable N-terminal part of the receptor, designated as AF These have helped significantly in understanding the molecular aspects of DNA and ligand binding, but have to some extent also led to biased attention to these parts of the receptor only.
For example, many coregulator interaction studies are still performed with the LBD only, while numerous studies have demonstrated that the AF-2 domain often tells only part of the story. With the help of biophysical techniques, however, it is feasible to study the full-length receptor in its native environment Figure 2. Upon entering the cell by passive diffusion, the hormone H binds the receptor, which is subsequently released from heat shock proteins, and translocates to the nucleus.
There, the receptor dimerizes, binds specific sequences in the DNA, called Hormone Responsive Elements or HREs, and recruits a number of coregulators that facilitate gene transcription. This latter step can be modulated by receptor antagonists like tamoxifen T , and cellular signalling pathways.
Examples of processes studied using biophysical techniques and discussed in this review include: Steroid Hormones SHs reach their target cells via the blood, where they are bound to carrier proteins. Because of their lipophilic nature it is thought that they pass the cell membrane by simple diffusion, although some evidence exists that they can also be actively taken up by endocytosis of carrier protein bound hormones [ Hammes et al. While this is likely the case for typical agonists like estrogen and progesterone, this is not always correct for receptor antagonists.
These antagonists come in two kinds, so-called partial antagonists for the estrogen receptors known as SERMs for Selective Estrogen Receptor Modulators and full antagonists. The partial antagonist can, depending on cell type, act as a SHR agonist or antagonist. In contrast, full antagonists for ER known as SERDs for Selective Estrogen Receptor Downregulators always inhibit the receptor, independent of cell type, in part by targeting the receptor for degradation. Binding of either type of antagonist results in major conformational changes within the LBD and in release from heat shock proteins that thus far had protected the unliganded receptor from unfolding and aggregation Figure 2 , step 2.
This process was nicely visualized for the estrogen receptor by Devin-Leclerc et al. Although family members, SHRs are located differently in cells. The subcellular localisation of SHRs in living cells has been extensively studied using fusion constructs of green fluorescent protein GFP.
This showed that SHRs can be divided in three groups based on their unliganded distribution: The progesterone receptor is of particular interest as it exists in two forms with different ratios of nuclear versus cytoplasmic localization of the unliganded receptor. In most cell contexts, the PRA isoform is a repressor of the shorter PRB isoform, and without hormone induction it is mostly located in the nucleus, whereas PRB distributes both in the nucleus and in the cytoplasm.
PRB accumulates in the nucleus after progesterone binding, a process that directly correlates with PR mediated transcription [ Li et al. Rapid and almost complete nuclear translocation following ligand addition is a common behavior observed for almost all SHRs Figure 2 , step 3. This translocation coincides with a striking alteration in receptor distribution within the nucleus, most apparent in the case of the already nuclear ER.
Other groups confirmed similar behavior for the other SHRs, some directly by tagging two receptors with different variants of GFP and following both at the same time [ Nishi et al. Fejes-Toth and colleagues demonstrated that hormone-activated MRs accumulated in dynamic, discrete clusters in the cell nucleus, a phenomenon that only concurred with agonistic mineralocorticoids and not with full antagonists [ Fejes-Toth et al.
The exact nature of these foci is still unclear and multiple roles have been proposed, including storage depots and sites of transcription, splicing, aggregation or degradation. What is clear, however, is that nuclear and subnuclear translocation of SHRs is ligand and concentration dependent. Instead of a constitutive nuclear localization, this chimeric receptor adapted the cytoplasmic localization of unliganded GR, and translocates to the nucleus upon ER ligand addition.
Interestingly, the GR-ER chimera retained the anti- estrogen binding properties, and could thus be used to screen for new ER ligands.
Nuclear receptors that bind steroid hormones typically form homodimers Figure 2 , step 4. Dimer formation is facilitated mainly through interactions between the LBDs of both receptors, and is essential for their function, as mutations in the dimerization domain typically render the receptor inactive.
ER has been reported to exist as a dimer even in the absence of ligand, although it is important to note that these studies have again been performed with the LBD domain only [ Salomonsson et al. Biophysical in vitro studies, again with the LBD only, have confirmed these data and show slow dissociation of unliganded dimers, which is further retarded by ligand binding [ Tamrazi et al.
Recent in vivo studies suggested that this might not hold for full-length receptors though, at least not for AR. Importantly, FRET is extremely sensitive to the distance between the fluorophores its efficiency decays with the distance to the sixth power , and will therefore only occur when two proteins are on average no more than one molecule in distance apart, but usually they interact directly Figure 3 A.
Further study is required to confirm these findings and to determine whether this behavior is unique for AR or also applies to other SHRs.
Principle of FRET to measure intermolecular interaction. This phenomenon only occurs when both monomers physically interact as a dimer, and results in increased YFP emission at nm at the cost of CFP emission at nm. A similar protocol is followed to measure intramolecular FRET. Ligand binding induces conformational changes within the receptor and alters the relative orientation and distance between the two fluorophores, leading to changes in FRET efficiency.
Fluorescence is depicted in false colors. The fluorescent signal of YFP yellow and CFP cyan is arbitrarily set to the same level such that the ratio red is 1, and followed in time. After 80 seconds, tamoxifen is added to the cells arrowhead and a conformational change is observed in the form of a change in FRET; the YFP signal increases at the cost of CFP fluorescence.
Dimers of SHRs are only efficiently formed between closely related receptors. In this light the previously mentioned two isoforms of the progesterone receptor and the two estrogen receptors are of particular interest. In both cases one of the two seems to exhibit a repressive function on the other. 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.
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. 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 Figure 4. 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.
The nature of the substrate on which the receptor immobilizes remains uncertain, but almost certainly includes DNA. Upon ligand binding, the residence time on DNA is significantly increased. According to Farla et al. The fluorescence recovers over time by diffusion. 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. 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. Interestingly, FRAP analysis on the array again shows a rapid exchange of receptors between chromatin and the nucleoplasmic compartment.
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.
Strikingly, addition of a full-antagonist showed the opposite effect, with ongoing exchange at a rate faster than for an agonist bound receptor.
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.
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. Recently a number of groups have claimed a role for SHRs in non-genomic, extranuclear signalling events Figure 2 , step 6.
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. For transcription to occur the subsequent recruitment of coregulator proteins is absolutely required Figure 2 , step 7. These regulatory proteins come in two types, coactivators and corepressors that respectively enhance or diminish transactivation activity through various enzymatic activities, including acetylating, deacetylating, methylating, ubiquitinating, and kinase activity.
Ligand dependent recruitment of coregulators occurs through a hydrophobic cleft formed by helices 3, 4 and 12 in the AF-2 domain of the receptor [ Gronemeyer et al. Upon hormone binding, this helix is repositioned, which opens a functional interface for coregulator recruitment through conserved LXXLL motifs in the cofactor.
Antagonists exert their function by inducing a different conformational change of H12 that blocks or modulates the recruitment of these essential coregulators. However, not all coregulator binding occurs through the AF-2 region. Other conformational changes within the receptor and events like dimerization are also likely to be involved in coregulator recruitment.
Moreover, the AF-1 region of SHRs plays an important role in ligand independent binding of coregulators. The exact coregulator requirements for transcription are dependent on cell type, and probably also on ligand and promoter context. This explains why partial antagonists can have antagonistic properties in one tissue, while exhibiting agonistic properties in another. A large number of publications have since confirmed these findings, also with full-length ER constructs.