N-terminal Domain Regulates Steroid Activation of Elephant Shark Glucocorticoid and Mineralocorticoid Receptors

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Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD+LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR.

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  1. ###Reviewer #3

    This is a straightforward study addressing the evolutionary divergence of the glucocorticoid receptor. Authors use the GR and MR receptors from elephant shark which represent distinct orthologs of human GR/MR which diverged from a common CR receptor in cartilaginous fishes. The authors address two functional questions regarding 1) agonist/antagonist specificity between ES GR/MR and 2) The functional role of the AB domain (N terminal domain) of the GR/MR which is known to play a specific role in GR transactivation. The study is technically well executed. However, the following should be addressed.

    1. While the introduction is informative, it is difficult to follow as the authors describe ligand activities for two receptors, multiple ligands, multiple species and chimeras. While this information is summarized well in Table 1, it does not appear until well into the manuscript. It would help readers, I believe, to be more general in the introduction rather than provide a plethora of ligand specificities.

    2. Given that a large component of the study focuses on the functionality of the GR A/B (AF1) activation domain here termed the "NTD" it would seem prudent to have some introduction and/or discussion on the role of this domain in NR's in general. Depending upon which of the NRs is being addressed the AB domain may serve multiple functionalities. For instance, the AB domain domain is a target site for receptor phosphorylation through differing kinase/phosphatase activities. Phosphorylation within the AF-1 domain can significantly affect transcriptional activity and impact ligand dependent and ligand independent activities. For example, Estrogen receptors are phosphorylated at both serine and threonine residues by mitogen activated kinase (MAPK) following growth factor stimulation and enhance transcriptional activity. PPAR and PPAR are additionally phosphorylated within the A/B domain yet exhibit reciprocal transcriptional activation (PPAR) and repression (PPAR). VDR and RXR also have putative phosphorylation sites. In this study, no mention is given to the role of Ab domain phosphorylation or how the functionality of the AB domain might be involved in allosteric interactions that facilitate ligand receptor binding.

    3. As stated in comment 2 above, the study could also be greatly enhanced if the authors further conducted experiments to further refine the region of importance within the NTD that facilitates the activation of the GR. Alignment of the two sequences could help infer potential targets and functional mutation studies may provide greater mechanistic insight into the NR functionality and aid making evolutionary inferences in how MR and GR have diverged from human GR/MR. This aspect of the study could also be modeled using a three-dimensional molecular docking approach.

    4. The experiments were conducted in HEK cells, which may or may not contain essential coregulators necessary for driving transactivation. It is also highly noticeable that MR activities are significantly attenuated compared to GR. Comment by the authors on both these points would be useful.

    5. While the study recognizes the significant differences in EC50 across receptor types and their ligands, little attention is given to the Emax for each of the assays. It seems strange that ES-MR demonstrates a great potency for cortisol and corticosterone than GR however the Emax values for GR are magnitudes greater than MR.

    Minor comments:

    Why was the AF2 domain left out of Figure 2?

  2. ###Reviewer #2

    The authors report the first characterization of the elephant shark glucocorticoid receptor (GR). In my view, the experiments are a useful contribution to the literature, but the significance of the work as presented is limited.

    They have two new findings:

    1. The elephant shark GR does not activate in response to progesterone or 19norP, despite the steroids binding to the GR. This contrasts with their previously published characterization of the elephant shark MR (ref #36). GR from other organisms does not activate with progestins, but also does not bind them.

    2. The GR N-terminal domain (NTD) dramatically increases the fold activation of the GR, but has no apparent effect on steroid specificity (Figure 4). This is a property of the NTD, as swapping the GR NTD onto the MR ligand-binding domain leads to elevated activity (Figure 6). This behavior matches what has been seen previously for bony-vertebrate GRs, but has not been demonstrated for cartilaginous fish GRs.

    They have one finding that is somewhere between new and confirmatory:

    1. The elephant shark GR behaves similarly to the previously characterized skate GR (refs #7, #37) in that it responds to both aldosterone and cortisol and has lower sensitivity to steroids than the MR.


    I found this paper difficult to read. The introduction was long. It was difficult to tell from the introduction what was previously known and what was new in this paper. The results had little narrative structure, making it difficult to understand why the authors chose to do each experiment. And the discussion did not really explore the implications of their observations.

    There are four data figures and a table. Of those, Figs 3, 4, and Table 1 are the same data shown in different ways. Of the data in these figures, the MR bits-about half of the data-have already been published for slightly different constructs of the same proteins (ref #36). The work was observational, with no mechanism – evolutionary, biochemical, physiological, or otherwise – presented.

    Specific comments:

    1. The authors never discuss the implications of their results for the physiology of elephant sharks. Why should it matter that the elephant shark GR does not respond to progesterone and 19norP? Is this surprising given what we know about GRs from other species?

    2. The authors don't "close the loop" on their evolutionary questions regarding steroid specificity. How does their work contribute to our understanding of the evolution of the GR and its function across vertebrates? Can they propose when the progesterone response evolved (or was lost)? Was it gained on the MR lineage or lost on the GR lineage? (One of the papers the authors cite (Bridgham et al, #7) reports that the hagfish CR-which is co-orthologous to MR and GR from jawed vertebrates-responds to progesterone. It seems like this is worth bringing into their discussion). In general, a much more fleshed out discussion of what is known about GR and MR from other cartilaginous, ray-finned and jawless fishes is in order.

    3. The authors argue that the importance of the NTD for GR activation, but not MR activation, indicates that the NTD activity evolved after the divergence of MR and GR. It is equally likely, however, that MR NTD lost its ancestral ability to activate. (This could be tested by, for example, characterizing full-length CR from hagfish or lamprey and asking if its NTD is more MR-like or GR-like in function.)

    4. In the paragraph starting “Activation of elephant shark GR by aldosterone...”, the authors should probably note that the previously characterized skate GR responds to aldosterone and cortisol, as does the reconstructed ancestor of GR and MR (refs #7, #37). This adds heft to their claim that GR is transitional from MR in elephant shark.

    5. The authors motivate their decision to characterize the full-length elephant shark GR by saying that because no full-length elasmobranch GR has been characterized, "the identity of the physiological glucocorticoids in cartilaginous fish is not known." This seems odd, given that, to a first approximation, most GR NTDs amplify the response to all steroids without dramatically altering specificity (see, Figure 4A and C, for example). Is there some reason the authors expect the NTD to alter specificity in this case? Further, all of the data in this manuscript are in vitro: this cannot show whether these steroids are physiological or not.

    Minor comments:

    In several places, the language the authors chose seems to imply that the elephant shark is ancestral. The sentences should be modified to indicate that the shark gives insight into an ancestral state, but is not itself ancestral.

  3. ###Reviewer #1

    This is a well carried out study of the ligand specificity and also the role of the NTD of elephant shark GR and MR. The study though, conflates two things – the role of the NTD in transactivation (it is well known that the NTD of steroid receptors contains a transcription activation function – TAF1) – and the role of the NTD in ligand binding (allosteric interactions between the NTD and the ligand binding domain; LBD). While it is possible that the NTD exerts an allosteric influence over the LBD, as suggested by the authors, I do not feel that this conclusion is justified by the data presented.

    Major points:

    1. The introduction conflates the two issues of allosteric interaction between NTD and LBD (e.g., as shown in ref 22) with the existence of a TAF in the NTD (demonstrated in ref 19 for example). The activation domain is autonomous, requiring tethering to DNA by the DNA binding domain of the receptor. This applies to the statement “It is not known when the strong dependence of vertebrate GR on the NTD for activation of gene transcription evolved”. However, it has also been demonstrated (though not in most of the references cited) that there is an allosteric interaction by which the GR NTD alters ligand binding (i.e., affinity) by the LBD, alluded to (albeit not explicitly) earlier on.

    This is problematic when it comes to the way the data in Figure 3 are described. Activation of a reporter gene was measured, not activation of the receptor. If the reporter gene had not contained a GRE, there would have been no effect of steroid on the experimental readout, but the receptor would still have been activated by the steroid. What Figure 3 shows is that the GR NTD contains a strong transcriptional activation domain, required for induction of MMTV LTR-luciferase, consistent with previously published data. However, the MR does not have a TAF in the NTD (at least one active at this promoter); deletion of the NTD has no effect on transactivation of the reporter. The data in Figure 3 say nothing about the affinity of the receptors for the ligands. To infer anything about ligand-dependent activation of receptor, a ligand dose-response is required (as in Figure 4).

    1. It is not clear if the EC50s reported in Table 1 are sufficiently (significantly) different to each other in order to infer anything about the influence of the NTD on ligand binding. No statistical analysis has been performed on the EC50s.

    The similarities of the EC50s for all 4 corticosteroids for the truncated and full-length MR supports the EC50 being determined by the LBD and is consistent with no role for TAF1 at this promoter. For the GR, the EC50s reported in Table 1 derive from the data shown in Fig4. There are very minor differences in EC50 for corticosterone (the highest affinity ligand) between GR-FL, GR-truncated and the MR-GR chimera. This suggests the affinity of the receptor for this ligand is determined by the LBD. Likewise, the affinity of all 3 receptors for cortisol is within experimental error (with the additional caveat that the graphs in Figure 4 do not plateau for cortisol, so the estimate of EC50 is likely to be inaccurate; this caveat also applies to the statement “the EC50 for cortisol increased over 2-fold, and an EC50 for 11-deoxycorticosterone was too low to be calculated”). Aldo also doesn't reach plateau, so the same caveats apply there (and there is <3-fold difference between the EC50 for GR-FL and MR-GR). This I do not feel that the data support the conclusion drawn regarding allosteric signalling between NTD and LBD (“These results indicate that allosteric signaling between the NTD and DBD-LBD in elephant shark GR is critical for its response to corticosteroids, in contrast to elephant shark MR”).

    1. The same issue arises in the description of the results of the chimera experiment (starting “Thus, in the GR NTD-MR DBD-LBD chimera...”). The reason that fusion of the GR NTD to MR LBD conferred greater activation of the reporter gene is because it fused a strong activation domain (TAF1) to the MR LBD. This also allowed the prog and 19norprog activation - the fusion of the GR TAF1 to the MR ligand specificity. This was to be expected. Similarly, the MR NTD does not contain a TAF (at least one active at this particular promoter). So it was only to be expected that a fusion of the MR NTD to GR LBD would lack the strong GR TAF.

    Minor comments:

    The authors might want to discuss RU486 (which binds to both GR and PR) with respect to the experiments shown in Figure 5.

  4. ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to Version 1 of the preprint: https://www.biorxiv.org/content/10.1101/822718v1.full


    This paper uses the sequences of and experiments involving the mineralocorticoid (MR) and glucocorticoid (GR) receptors from a cartilaginous fish (the elephant shark), a sister taxa to the ray-finned fish and terrestrial vertebrates, to investigate the early evolution of the specificity of these important steroid receptors.

    The reviewers appreciate the value of studying the activity and specificity of steroid receptors (SR) from a taxon that diverged from its common ancestor with vertebrates close to 500 million years ago, but identified several important issues that they feel limit the impact of the manuscript. These are described in detail in the individual reviews.

    1. In the interpretation of their experiments on the N-terminal domain (NTD), the authors conflate two things: the role of the NTD in transactivation and its role in ligand binding. This leads to a conclusion – that there is an allosteric interaction between the NTD and the ligand binding domain (LBD) – this is not demonstrated.

    2. The in vitro characterization of the activity and steroid specificity of elephant shark SRs in an in vitro assay is a useful contribution. However, in the absence of a stronger relationship of these experimental observations to a biochemical mechanism of action, a specific evolutionary scenario, or to elephant shark physiology, the broader significance of these findings is unclear.

    3. Some of the statistical analyses and evolutionary analyses need stronger support.