BACKGROUND
the nuclear receptor super-family of proteins includes steroid receptors that are ligand-activated transcription factors mediating steroid actions on a multitude of behavioral, morphological and physiological processes  <cit> .
the two major functional groups of vertebrate corticosteroid receptors are glucocorticoid  and mineralocorticoid  receptors distinguished by their amino acid sequences and ligand specificity.
jawless vertebrates  have a single corticosteroid receptor, while jawed cartilaginous and bony fishes possess grs and mrs  <cit> .
bony fishes fall into two main clades: lobe-finned that includes tetrapods and ray-finned that includes teleosts, the most species-rich group of vertebrates  <cit> .
functional relationships between the grs and mrs of tetrapods and teleosts remain somewhat uncertain due, in part, to a lack of consensus as to the bioactive mineralocorticoid ligand and the presence of two grs in some teleosts.
here, we compare the ligand specificity of gr and mr ligand binding domains  between mammals and teleosts, presenting new evidence to support the hypothesis that neo-functionalization likely facilitated the maintenance of duplicate grs in teleosts following an initial duplication event with the origin of bony fishes.
focusing on the lbd allowed us to compare variation in ligand binding without the confounding effects of variation in other domains, such as the dna binding domain  and n-terminal region that can alter transactivational activity  <cit> .
among most mammals, including humans, cortisol is the principal glucocorticoid that induces the transcriptional activities of both the gr and mr  <cit> .
the mineralocorticoid aldosterone also activates mr, but not gr  <cit> .
like mammals, cortisol is the major glucocorticoid among teleosts  <cit> .
however, unlike mammals, there is no convincing evidence for significant circulating levels of aldosterone among teleosts  <cit> ; the aldosterone precursor 11-deoxycorticosterone  is a prominent candidate for the dominant mineralocorticoid in teleosts  <cit> .
most teleosts have two grs  <cit> , with some being cortisol-specific and others activated in vitro by both aldosterone and cortisol in some species  <cit> .
the existence of teleost grs with aldosterone sensitivity, but where cortisol may be the predominant ligand in the absence of aldosterone, has prompted investigations like the current one into the functional and evolutionary relationships of corticosteroid receptors and the ligands that induce their transcriptional activation.
to rigorously investigate the functional diversity of corticosteroid receptors, we compared the dose-dependent ligand sensitivity of the lbd of mrs and grs between a teleost, the midshipman   <cit> , and a mammal, the mouse .
midshipman fish are a key model for steroid modulation of reproductive behavior at multiple levels of analysis ranging from behavioral and molecular neuroendocrinology to neurophysiology  <cit> .
inclusion of the midshipman, from which a partial gr  and partial mr sequences have been published  <cit> , and for which we report a second gr  here, also provided us the opportunity to compare ligand specificities of mrs and grs between distantly related teleosts using a single assay.
to this end, we examined aldosterone and cortisol activation of mr, gr <dig>  and gr <dig> from a cichlid .
cichlids are a representative of the largest order of teleosts   <cit> , have duplicate grs  <cit> , and are another teleost model for the behavioral neuroendocrinology of reproductive plasticity  <cit> .
since multiple domains of steroid receptor gene products can influence transcriptional activity  <cit> , we cloned the lbd of each receptor in frame with the dna binding domain  of the yeast transcription factor gal <dig>  we employed a gal4-uas promoter to control expression of a luciferase reporter gene, similar to previous studies in heterologous cell systems  <cit> , as well as in vivo  <cit> .
this system allowed us to focus on variation in ligand binding independent of variation in other properties of the receptor .
using a battery of up to seven steroids, we show differences in the lbd specificity of duplicate teleost grs, as well as the grs between teleosts and mammals.
when viewed broadly in the context of a phylogenetic analysis of corticosteroid receptors, the results suggest that duplicate grs among teleosts exhibit an evolutionarily labile pattern of ligand binding that is unique among the major vertebrate lineages with one glucocorticoid-specific gr and another gr with more general corticosteroid sensitivity, similar to the ancestral vertebrate mr.
RESULTS
phylogenetic analysis of corticosteroid receptors
to more completely characterize the evolutionary relationships between the corticosteroid receptors of our main study species and other vertebrates, we first cloned and sequenced the full lbd with the hinge region and a short portion of the dbds of midshipman mr  and gr <dig> .
the resulting sequences were aligned with multiple corticosteroid receptors to verify the identity and similarity of the cloned genes with their homologs from other species.
the cloned regions of the corticosteroid receptors included the entire coding region for the lbd, hinge region, and a few amino acids of the dbd sequence.
a phylogram of corticosteroid receptor sequences had 100% posterior probabilities at each node .
due to a discrepancy between the names and the phylogenetic relationship of burton's cichlid gr <dig> and gr <dig> relative to other teleosts  <cit> , a recent report renamed their grs  <cit> .
we utilized this more recent and phylogenetically consistent naming system.
as suggested previously  <cit> , the results indicated that the ancestral corticosteroid receptor  underwent a duplication event giving rise to gr and mr prior to the divergence of cartilaginous  and bony fishes that includes sarcopterygians/lobe-finned fish that gave rise to tetrapods .
a second duplication event resulted in gr <dig> and gr <dig> within the second major clade of bony fish, the actinopterygian/ray-finned fishes .
ligand specificity
using a heterologous expression system, we tested the activity of corticosteroid receptor homologues induced by a variety of steroid hormones found among vertebrates.
to do this we first confirmed expression of the full-length lbd-gal <dig> fusion products from the plasmid constructs by western blotting, which showed bands of the expected sizes for each of the constructs .
as an initial screen for ligand specificity and efficacy of the expression system, we measured the luciferase activity produced by cells co-transfected with each of the lbd-gal <dig> constructs and the luciferase reporter construct in response to hormone treatments at one concentration, 10- <dig> m. normalized values were expressed as fold change over vehicle  treatment .
significant ligand induced activities were observed for all the receptor lbds examined.
the mr constructs from both midshipman and mouse showed significant activation in response to 10- <dig> m concentrations of aldosterone and cortisol .
the cortisol precursor 11-deoxycortisol  also produced activity significantly above baseline .
mouse gr lbd was sensitive only to cortisol and the synthetic glucocorticoid dexamethasone but not to the mineralocorticoid aldosterone , consistent with previous results from rats  <cit> .
midshipman gr <dig>  behaved similar to mouse gr, with significant activity induced only by cortisol and dexamethasone.
midshipman gr <dig> was also activated by cortisol and dexamethasone, but like mouse and midshipman mrs showed significant activity in response to 10- <dig> m of aldosterone .
the results of this initial screening demonstrated that:  the lbds were sufficient to confer ligand specificity,  the grs showed strong specificity for cortisol over its upstream and downstream biosynthetic products 11-deoxycortisol and cortisone , and  aldosterone sensitivity was variable among the grs, found only for gr <dig>
ligand sensitivity
to rigorously characterize the ligand sensitivity of the receptors, we performed dose response experiments with the lbd constructs from the mrs and grs of the mouse and midshipman using cortisol, doc and aldosterone as well as dexamethasone for the grs .
as noted earlier, doc was included because it has been suggested that this steroid, a precursor to aldosterone in tetrapods, may function as an important agonist of mrs in teleost fish that lack aldosterone  <cit> .
we estimated ec <dig> values  from the logistic dose response curves shown in figure  <dig>  for several of the responses here, such as midshipman mr with cortisol, receptor activation initially increased and then decreased with increasing ligand doses, a trend previously observed in corticosteroid receptors .
this may be indicative of cytotoxicity at high steroid doses  <cit> .
this reduced activity at higher doses leads to underestimated upper bounds in the fitted logistic curves that, in turn, yield underestimates of the corresponding ec <dig> values.
to counter this effect when performing our logistic fits, we excluded doses at which the observed activity decreased relative to lower doses .
note: ec <dig> ligand nanomolar concentrations based on logistic regressions.
no response is indicated by: --.
nd indicates that the ec <dig> could not be determined based on the curve.
as in the initial screen, mr lbds were sensitive to both aldosterone and cortisol .
the ec <dig> of cortisol was an order of magnitude higher than that of aldosterone for the mouse mr, while the ec50s for aldosterone and cortisol were approximately the same as for midshipman mr .
however, the maximum fold activation induced by aldosterone was nearly twice that induced by cortisol for midshipman mr .
therefore, while the ec50s of cortisol and aldosterone did not differ substantially, the level of activity induced at the ec <dig> was much higher with aldosterone.
doc also induced activity of both mouse and midshipman mr constructs.
the dose response curve for doc in midshipman was very similar to that of cortisol .
doc did not activate mouse mr to the same maximal level as either aldosterone or cortisol and the ec <dig> of doc, like that of cortisol, was approximately an order of magnitude higher than that of aldosterone.
the gr lbd constructs were most sensitive to dexamethasone , as reported previously for full length grs  <cit> .
this high dexamethasone sensitivity was evident in both the maximum observed activation as well as the ec <dig> values.
for all grs, the ec <dig> for cortisol was approximately an order of magnitude, or more, greater than that of dexamethasone.
the response to cortisol was fairly similar across grs, with a somewhat lower ec <dig> for mouse gr than either midshipman grs.
the largest distinction between the gr constructs was in the activation by aldosterone and doc.
while mouse gr was not activated by aldosterone in our initial screen using 10­ <dig> m aldosterone , 10­ <dig> m aldosterone caused substantial activation  as seen previously for rat gr  <cit> .
doc, however, failed to activate this receptor at any concentration.
the midshipman gr <dig> construct showed no response to either aldosterone or doc at any of the doses examined .
in contrast to midshipman gr <dig>  midshipman gr <dig> was sensitive to both aldosterone and doc .
neither of these ligands induced the levels of response in gr <dig> that we observed with either dexamethasone or cortisol.
the sensitivity of the gr <dig> construct to low doses of aldosterone and doc made accurate ec <dig> estimates impossible .
none of the lbd constructs were activated by either cortisone or 11-kt , indicating that the low sensitivity of the gr <dig> construct to aldosterone and doc did not reflect this artificial construct's overall lack of specificity.
rather, this likely reflected true mineralocorticoid sensitivity of midshipman gr <dig>
gal4-lbd assays, like those used here and in other studies  <cit> , allowed us to assess variation in lbd activation, while eliminating differences in activity that may result from variation in the n-terminal a/b domains or dbds  <cit> .
though the remainder of the receptor may modify ligand responses, our results using gal4-lbd constructs likely reflected the activity of the full-length receptor .
amino acid substitutions and gr ligand specificity
activation of the duplicate grs by doc and/or aldosterone was not consistent with the phylogenetic relationships of the receptors .
for example, the aldosterone sensitivity of gr <dig> and of gr <dig> varied between cichlid species .
this suggested that mineralocorticoid sensitivity was evolutionarily plastic and may have resulted from a small number of amino acid substitutions in the grs rather than overall sequence homology.
strong candidates for amino acids regulating mineralocorticoid sensitivity are those that are conserved between grs with similar mineralocorticoid sensitivities, but not between grs with differing sensitivities, independent of phylogeny.
we compared the mineralocorticoid-sensitive gr lbd sequences from burton's cichlid gr <dig> and midshipman gr <dig> with the mineralocorticoid-insensitive rainbow trout gr <dig> and midshipman gr <dig> .
nine amino acids were conserved within, but not between, these groups .
note: aldo, aldosterone; doc, 11-deoxycorticosterone; dex, dexamethasone; reichstein's compound s, 11-deoxycortisol; reichstein's compound e, cortisone.
+ and  <dig> indicate activation or lack of activation, respectively, by each ligand to induce transactivational activity at concentrations of ≤ 10- <dig> m. n.a.
indicates that the data are not available.
data from previous studies are indicated by the citations following the receptor type.
the mr response to dexamethasone is excluded because of the confounding dexamethasone paradox  <cit> .
1activity at >10- <dig> m;
2disagreement between previous studies;
3possible low activation at 10- <dig> m
to test the role of the nine candidate amino acids on gr mineralocorticoid sensitivity, we performed transactivation assays using gal <dig> constructs of daffodil cichlid mr, gr <dig>  and gr <dig> with 10- <dig> m aldosterone and cortisol .
at the nine amino acid positions identified above, the lbd of daffodil cichlid gr <dig> was found to be identical to midshipman gr <dig> while daffodil cichlid gr <dig> was identical to midshipman gr <dig>  the daffodil cichlid mr construct was activated by both aldosterone and cortisol, consistent with midshipman mr and a previous study of full-length burton's cichlid mr  <cit> .
the daffodil cichlid gr <dig> construct was activated by both aldosterone and cortisol, while daffodil cichlid gr <dig> was cortisol-specific at 10-7m, consistent with a role of the identified amino acid substitutions in regulating mineralocorticoid sensitivity.
we next used protein structure homology modeling to examine the locations of the identified amino acid substitutions and access the likelihood that such substitutions might alter ligand specificity.
modeling the midshipman gr lbds was based on those of a putative ancestral gr and a general corticosteroid receptor.
two of the nine identified candidate amino acids, a/s <dig> and f/i <dig>  appeared to be particularly strong candidates for regulating lbd function due to estimated proximity to the bound corticosteroid or to structural change induced in the protein models .
the a/s <dig> substitution was located at the end of helix  <dig>  distant from the ligand-binding pocket; however, this substitution between polar and non-polar residues altered the length of helix  <dig> and the loop between helices  <dig> and  <dig> .
this may have an important effect on the relative positioning of helix  <dig> and thus how well the ligand-binding pocket can accommodate either aldosterone or doc.
furthermore, the side chains of the residues of substitution f/i <dig> are located less than  <dig> Å from the putative bound ligand position .
this substitution represents a major change in the physical structure of the ligand-binding pocket of the lbd, as well as a change in the degree of hydrophobicity.
such a change could alter the positioning and the binding of a ligand within the ligand-binding pocket.
the seven remaining amino acid substitutions were more distant from the ligand-binding pocket and failed to induce structural changes between the homology models.
discussion
we took a broad comparative approach using chimeric gal4-lbd constructs in a single assay to examine the ligand specificity of the lbds of the full range of corticosteroid receptors within single species as well as across deeply diverged vertebrate taxa.
as discussed below, this property likely led to neo-functionalization and maintenance of two grs.
comparisons of the amino acid sequences and predicted structures of the duplicate gr lbds added insight into the evolutionary changes that may regulate ligand specificity.
in particular, the mineralocorticoid sensitivities of duplicate grs in teleosts were highly labile in the context of their molecular phylogeny, i.e., it could not be predicted based on the degree of similarity between their sequences .
ligand specificity
aldosterone is the primary mineralocorticoid in tetrapods  <cit> , while teleost fish appear to lack the aldosterone synthase necessary to produce aldosterone  <cit> .
we demonstrated that teleost mr lbd is sensitive to both mineralocorticoids and glucocorticoids, consistent with previous results for both the lbd and the whole protein  <cit> .
the maximal activation by aldosterone was greater than that of either doc or cortisol , though the variation in ec <dig> values of these ligands for the midshipman mr construct were not substantial .
doc, the potential natural agonist for mr in teleosts  <cit> , elicited a response from midshipman mr. this was consistent with results for the full-length mr of rainbow trout  <cit> , the mr lbd of skate  <cit> , and the corticosteroid receptors of the more basal hagfish and lamprey   <cit> .
our data thus support the hypothesis that the evolution of an mr predates aldosterone  <cit> .
doc, which elicits activity from mrs across a range of vertebrates , may be the primary ligand of the ancestral mr  <cit> .
since doc activates the corticosteroid receptors of jawless vertebrates  <cit> , the proposed ancestral corticosteroid receptor   <cit> , the maintained doc sensitivity of mr may represent a conserved ancestral function.
aldosterone induced greater maximal activation than doc for both mouse and midshipman mr lbd constructs with lower ec50s, suggesting that aldosterone evolved as a more potent mr agonist among lobe-finned fishes, as evidenced in extant lungfish and descendant tetrapods  <cit> .
the response of the mouse mr to doc , together with evidence for doc in the plasma of mammals , suggests that doc may still be an important ligand of the tetrapod mr. the mr lbds studied here showed sensitivity to both aldosterone and cortisol, as well as two steroids involved in the biosynthesis of aldosterone and cortisol  .
this reflects both the overall promiscuity of the mr and conserved sensitivity of the ancestral corticosteroid receptor .
three of these steroids  are present in the plasma of teleosts  <cit> ; thus, teleost mr has either a wide array of endogenous ligands or alternate mechanisms inhibit certain steroids from binding to mr, as occurs in mammals  <cit> .
neo-functionalization of duplicate grs
the relative ligand sensitivities of gr <dig> and gr <dig> in teleosts provide insight into the biological basis for maintaining duplicate grs.
bury and sturm  <cit>  hypothesized that the difference in sensitivity between rainbow trout grs evolved such that gr <dig> is active at basal cortisol levels while gr <dig> is recruited under stressful conditions with elevated cortisol.
the gr lbds examined in this study were all activated by cortisol and with a slightly lower ec <dig> for midshipman gr <dig> than gr <dig> , though not on the order of the difference observed in rainbow trout  <cit> .
the dose-response data showed activity from gr <dig> at very low cortisol concentrations, while gr <dig> was not activated at these concentrations .
this is further consistent with the hypothesis of gr <dig> activity restricted to times of high cortisol release.
bury and sturm  <cit>  described the observed difference in cortisol sensitivity between duplicate grs as a neo-functionalization and proposed that the functions of a single gr are partitioned between the duplicate grs into stressful  and basal-level  activity.
such partitioning seems more accurately described as sub-functionalization since neither of the duplicate grs acquire a novel function.
the evidence presented here supports neo-functionalization based on the promiscuity of ligand specificity among gr paralogues and suggests that promiscuous grs acquired a novel function distinct from their direct ancestral predecessor.
as we report for midshipman and daffodil cichlid, it appears that in many teleost species one gr shows strong ligand specificity for glucocorticoids , while the other gr responds to glucocorticoids as well as either aldosterone and doc or 11-deoxycortisol .
the broader ligand sensitivity is more similar to that of the single skate gr as well as the basal hagfish and lamprey corticosteroid receptors .
however, because the inferred ancestral gr of bony vertebrates is cortisol-specific  <cit> , the observed promiscuousness among grs represents a novel property and thus is a neo-functionalization, rather than sub-functionalization of the ancestral characters.
a similar reversion of gr to a more broadly sensitive receptor is seen in chickens  in which the full-length gr is more sensitive to aldosterone than to cortisol  <cit> .
a promiscuous gr among bony vertebrates could function in a role similar to the ancestral gr of cartilaginous fishes , while a cortisol-specific gr serves a more selective function.
the fact that gr steroid-specificity is not predictable based on the phylogeny of receptor sequences  suggests that activation by steroids other than cortisol is evolutionarily labile, providing a basis for rapid adaptation of these receptors.
the evolutionary plasticity of gr specificity may occasionally lead to the duplicate teleost grs reverting to redundant states and thus one of the receptors being lost in some lineages, which may be responsible for the single gr of zebrafish   <cit> .
amino acid substitutions and gr ligand specificity
we aligned the amino acid sequences of lbds from gr <dig> and gr <dig> of midshipman with gr <dig> from burton's cichlid and gr <dig> from rainbow trout to examine the molecular basis of variation in the mineralocorticoid sensitivity of grs.
nine amino acid positions correlated with mineralocorticoid sensitivity .
these positions showed the same correlation with mineralocorticoid sensitivity in the daffodil cichlid gr lbds , strongly supporting a role for these residues in altering ligand specificity.
five of the identified residues  are noteworthy because the same amino acids occur at these positions in the mineralocorticoid-sensitive chicken gr  <cit> , with residues h <dig> and f <dig> common in many mrs .
furthermore, residue f <dig> has previously been shown to directly interact with the ligand, is adjacent to another ligand contacting residue  <cit> , and is important for proper receptor co-activator and co-repressor binding  <cit> .
using homology model structures to midshipman gr <dig> and gr <dig>  we identified two of the nine amino acid residues as particularly strong candidates for regulating ligand specificity because they likely either make contact with the ligand or change the structure of the lbd .
the a/s substitution at position  <dig> is distant from the ligand-binding pocket, but it alters the length and polarity of the distal end of helix  <dig>  which is likely to affect the conformation and flexibility of helix  <dig> and the loop between helices  <dig> and  <dig>  such conformational effects can play an important role in the ligand specificity of corticosteroid receptors  <cit> .
additionally, the f/i substitution at position  <dig> is located in the binding pocket, within 4Å of the putative ligand position .
though this is a substitution between two non-polar residues, this substitution introduces a change in the degree of hydrophobicity and a large difference in structure.
such amino acid substitutions within the lbd affect the binding specificity and transactivational response of steroid receptors to particular ligands  <cit> .
our results are consistent with the findings of bridgham et al  <cit>  that accumulation of restrictive mutations make a reversal of protein structure and function unlikely.
none of the nine candidate amino acid residues identified in our analysis correspond to residues previously suggested to play a role in the evolution of gr cortisol specificity  <cit> .
this incongruity indicated that mineralocorticoid sensitivity among teleost grs likely results from novel mutations that permit aldosterone binding to otherwise cortisol specific receptors rather than back-mutations to the ancestral aldosterone sensitive state.
CONCLUSIONS
a luciferase reporter assay was used to characterize the transcriptional activation of corticosteroid receptors across the two most widely divergent groups of bony vertebrates, tetrapods and teleosts, showing the activation of all teleost receptors by cortisol with variable activation by other steroids in the corticosteroid biosynthetic pathways.
phylogenetic analyses demonstrated that gr aldosterone-sensitivity cannot be predicted among fishes on the basis of receptor sequence similarity.
this suggests that different lines of teleosts have adapted duplicate grs for divergent functions.
while one has cortisol-specificity, the other is broadly sensitive to corticosteroids, more akin to the ancestral state.
however, the latter is not a reversal of phenotype, but instead represents a novel condition brought about by specific amino acid substitutions.
such neo-functionalization reflects the dynamic and complex regulation inherent in the divergence of corticosteroid receptors across vertebrates.

 the nuclear receptor super-family of proteins includes steroid receptors that are ligand-activated transcription factors mediating steroid actions on a multitude of behavioral, morphological and physiological processes. the two major functional groups of vertebrate corticosteroid receptors are glucocorticoid and mineralocorticoid receptors distinguished by their amino acid sequences and ligand specificity. jawless vertebrates have a single corticosteroid receptor, while jawed cartilaginous and bony fishes possess grs and mrs. bony fishes fall into two main clades: lobe-finned that includes tetrapods and ray-finned that includes teleosts, the most species-rich group of vertebrates. functional relationships between the grs and mrs of tetrapods and teleosts remain somewhat uncertain due, in part, to a lack of consensus as to the bioactive mineralocorticoid ligand and the presence of two grs in some teleosts. here, we compare the ligand specificity of gr and mr ligand binding domains between mammals and teleosts, presenting new evidence to support the hypothesis that neo-functionalization likely facilitated the maintenance of duplicate grs in teleosts following an initial duplication event with the origin of bony fishes. focusing on the lbd allowed us to compare variation in ligand binding without the confounding effects of variation in other domains, such as the dna binding domain and n-terminal region that can alter transactivational activity. among most mammals, including humans, cortisol is the principal glucocorticoid that induces the transcriptional activities of both the gr and mr. the mineralocorticoid aldosterone also activates mr, but not gr. like mammals, cortisol is the major glucocorticoid among teleosts. however, unlike mammals, there is no convincing evidence for significant circulating levels of aldosterone among teleosts; the aldosterone precursor 11-deoxycorticosterone is a prominent candidate for the dominant mineralocorticoid in teleosts. most teleosts have two grs, with some being cortisol-specific and others activated in vitro by both aldosterone and cortisol in some species. the existence of teleost grs with aldosterone sensitivity, but where cortisol may be the predominant ligand in the absence of aldosterone, has prompted investigations like the current one into the functional and evolutionary relationships of corticosteroid receptors and the ligands that induce their transcriptional activation. to rigorously investigate the functional diversity of corticosteroid receptors, we compared the dose-dependent ligand sensitivity of the lbd of mrs and grs between a teleost, the midshipman, and a mammal, the mouse. midshipman fish are a key model for steroid modulation of reproductive behavior at multiple levels of analysis ranging from behavioral and molecular neuroendocrinology to neurophysiology. inclusion of the midshipman, from which a partial gr and partial mr sequences have been published, and for which we report a second gr here, also provided us the opportunity to compare ligand specificities of mrs and grs between distantly related teleosts using a single assay. to this end, we examined aldosterone and cortisol activation of mr, gr and gr from a cichlid. cichlids are a representative of the largest order of teleosts, have duplicate grs, and are another teleost model for the behavioral neuroendocrinology of reproductive plasticity. since multiple domains of steroid receptor gene products can influence transcriptional activity, we cloned the lbd of each receptor in frame with the dna binding domain of the yeast transcription factor gal we employed a gal4-uas promoter to control expression of a luciferase reporter gene, similar to previous studies in heterologous cell systems, as well as in vivo. this system allowed us to focus on variation in ligand binding independent of variation in other properties of the receptor. using a battery of up to seven steroids, we show differences in the lbd specificity of duplicate teleost grs, as well as the grs between teleosts and mammals. when viewed broadly in the context of a phylogenetic analysis of corticosteroid receptors, the results suggest that duplicate grs among teleosts exhibit an evolutionarily labile pattern of ligand binding that is unique among the major vertebrate lineages with one glucocorticoid-specific gr and another gr with more general corticosteroid sensitivity, similar to the ancestral vertebrate mr.  phylogenetic analysis of corticosteroid receptors to more completely characterize the evolutionary relationships between the corticosteroid receptors of our main study species and other vertebrates, we first cloned and sequenced the full lbd with the hinge region and a short portion of the dbds of midshipman mr and gr. the resulting sequences were aligned with multiple corticosteroid receptors to verify the identity and similarity of the cloned genes with their homologs from other species. the cloned regions of the corticosteroid receptors included the entire coding region for the lbd, hinge region, and a few amino acids of the dbd sequence. a phylogram of corticosteroid receptor sequences had 100% posterior probabilities at each node. due to a discrepancy between the names and the phylogenetic relationship of burton's cichlid gr and gr relative to other teleosts, a recent report renamed their grs. we utilized this more recent and phylogenetically consistent naming system. as suggested previously, the results indicated that the ancestral corticosteroid receptor underwent a duplication event giving rise to gr and mr prior to the divergence of cartilaginous and bony fishes that includes sarcopterygians/lobe-finned fish that gave rise to tetrapods. a second duplication event resulted in gr and gr within the second major clade of bony fish, the actinopterygian/ray-finned fishes. ligand specificity using a heterologous expression system, we tested the activity of corticosteroid receptor homologues induced by a variety of steroid hormones found among vertebrates. to do this we first confirmed expression of the full-length lbd-gal fusion products from the plasmid constructs by western blotting, which showed bands of the expected sizes for each of the constructs. as an initial screen for ligand specificity and efficacy of the expression system, we measured the luciferase activity produced by cells co-transfected with each of the lbd-gal constructs and the luciferase reporter construct in response to hormone treatments at one concentration, 10- m. normalized values were expressed as fold change over vehicle treatment. significant ligand induced activities were observed for all the receptor lbds examined. the mr constructs from both midshipman and mouse showed significant activation in response to 10- m concentrations of aldosterone and cortisol. the cortisol precursor 11-deoxycortisol also produced activity significantly above baseline. mouse gr lbd was sensitive only to cortisol and the synthetic glucocorticoid dexamethasone but not to the mineralocorticoid aldosterone, consistent with previous results from rats. midshipman gr behaved similar to mouse gr, with significant activity induced only by cortisol and dexamethasone. midshipman gr was also activated by cortisol and dexamethasone, but like mouse and midshipman mrs showed significant activity in response to 10- m of aldosterone. the results of this initial screening demonstrated that: the lbds were sufficient to confer ligand specificity, the grs showed strong specificity for cortisol over its upstream and downstream biosynthetic products 11-deoxycortisol and cortisone, and aldosterone sensitivity was variable among the grs, found only for gr ligand sensitivity to rigorously characterize the ligand sensitivity of the receptors, we performed dose response experiments with the lbd constructs from the mrs and grs of the mouse and midshipman using cortisol, doc and aldosterone as well as dexamethasone for the grs. as noted earlier, doc was included because it has been suggested that this steroid, a precursor to aldosterone in tetrapods, may function as an important agonist of mrs in teleost fish that lack aldosterone. we estimated ec values from the logistic dose response curves shown in figure for several of the responses here, such as midshipman mr with cortisol, receptor activation initially increased and then decreased with increasing ligand doses, a trend previously observed in corticosteroid receptors. this may be indicative of cytotoxicity at high steroid doses. this reduced activity at higher doses leads to underestimated upper bounds in the fitted logistic curves that, in turn, yield underestimates of the corresponding ec values. to counter this effect when performing our logistic fits, we excluded doses at which the observed activity decreased relative to lower doses. note: ec ligand nanomolar concentrations based on logistic regressions. no response is indicated by: --. nd indicates that the ec could not be determined based on the curve. as in the initial screen, mr lbds were sensitive to both aldosterone and cortisol. the ec of cortisol was an order of magnitude higher than that of aldosterone for the mouse mr, while the ec50s for aldosterone and cortisol were approximately the same as for midshipman mr. however, the maximum fold activation induced by aldosterone was nearly twice that induced by cortisol for midshipman mr. therefore, while the ec50s of cortisol and aldosterone did not differ substantially, the level of activity induced at the ec was much higher with aldosterone. doc also induced activity of both mouse and midshipman mr constructs. the dose response curve for doc in midshipman was very similar to that of cortisol. doc did not activate mouse mr to the same maximal level as either aldosterone or cortisol and the ec of doc, like that of cortisol, was approximately an order of magnitude higher than that of aldosterone. the gr lbd constructs were most sensitive to dexamethasone, as reported previously for full length grs. this high dexamethasone sensitivity was evident in both the maximum observed activation as well as the ec values. for all grs, the ec for cortisol was approximately an order of magnitude, or more, greater than that of dexamethasone. the response to cortisol was fairly similar across grs, with a somewhat lower ec for mouse gr than either midshipman grs. the largest distinction between the gr constructs was in the activation by aldosterone and doc. while mouse gr was not activated by aldosterone in our initial screen using 10­ m aldosterone, 10­ m aldosterone caused substantial activation as seen previously for rat gr. doc, however, failed to activate this receptor at any concentration. the midshipman gr construct showed no response to either aldosterone or doc at any of the doses examined. in contrast to midshipman gr midshipman gr was sensitive to both aldosterone and doc. neither of these ligands induced the levels of response in gr that we observed with either dexamethasone or cortisol. the sensitivity of the gr construct to low doses of aldosterone and doc made accurate ec estimates impossible. none of the lbd constructs were activated by either cortisone or 11-kt, indicating that the low sensitivity of the gr construct to aldosterone and doc did not reflect this artificial construct's overall lack of specificity. rather, this likely reflected true mineralocorticoid sensitivity of midshipman gr gal4-lbd assays, like those used here and in other studies, allowed us to assess variation in lbd activation, while eliminating differences in activity that may result from variation in the n-terminal a/b domains or dbds. though the remainder of the receptor may modify ligand responses, our results using gal4-lbd constructs likely reflected the activity of the full-length receptor. amino acid substitutions and gr ligand specificity activation of the duplicate grs by doc and/or aldosterone was not consistent with the phylogenetic relationships of the receptors. for example, the aldosterone sensitivity of gr and of gr varied between cichlid species. this suggested that mineralocorticoid sensitivity was evolutionarily plastic and may have resulted from a small number of amino acid substitutions in the grs rather than overall sequence homology. strong candidates for amino acids regulating mineralocorticoid sensitivity are those that are conserved between grs with similar mineralocorticoid sensitivities, but not between grs with differing sensitivities, independent of phylogeny. we compared the mineralocorticoid-sensitive gr lbd sequences from burton's cichlid gr and midshipman gr with the mineralocorticoid-insensitive rainbow trout gr and midshipman gr. nine amino acids were conserved within, but not between, these groups. note: aldo, aldosterone; doc, 11-deoxycorticosterone; dex, dexamethasone; reichstein's compound s, 11-deoxycortisol; reichstein's compound e, cortisone. + and indicate activation or lack of activation, respectively, by each ligand to induce transactivational activity at concentrations of ≤ 10- m. n.a. indicates that the data are not available. data from previous studies are indicated by the citations following the receptor type. the mr response to dexamethasone is excluded because of the confounding dexamethasone paradox. 1activity at >10- m; 2disagreement between previous studies; 3possible low activation at 10- m to test the role of the nine candidate amino acids on gr mineralocorticoid sensitivity, we performed transactivation assays using gal constructs of daffodil cichlid mr, gr and gr with 10- m aldosterone and cortisol. at the nine amino acid positions identified above, the lbd of daffodil cichlid gr was found to be identical to midshipman gr while daffodil cichlid gr was identical to midshipman gr the daffodil cichlid mr construct was activated by both aldosterone and cortisol, consistent with midshipman mr and a previous study of full-length burton's cichlid mr. the daffodil cichlid gr construct was activated by both aldosterone and cortisol, while daffodil cichlid gr was cortisol-specific at 10-7m, consistent with a role of the identified amino acid substitutions in regulating mineralocorticoid sensitivity. we next used protein structure homology modeling to examine the locations of the identified amino acid substitutions and access the likelihood that such substitutions might alter ligand specificity. modeling the midshipman gr lbds was based on those of a putative ancestral gr and a general corticosteroid receptor. two of the nine identified candidate amino acids, a/s and f/i appeared to be particularly strong candidates for regulating lbd function due to estimated proximity to the bound corticosteroid or to structural change induced in the protein models. the a/s substitution was located at the end of helix distant from the ligand-binding pocket; however, this substitution between polar and non-polar residues altered the length of helix and the loop between helices and. this may have an important effect on the relative positioning of helix and thus how well the ligand-binding pocket can accommodate either aldosterone or doc. furthermore, the side chains of the residues of substitution f/i are located less than Å from the putative bound ligand position. this substitution represents a major change in the physical structure of the ligand-binding pocket of the lbd, as well as a change in the degree of hydrophobicity. such a change could alter the positioning and the binding of a ligand within the ligand-binding pocket. the seven remaining amino acid substitutions were more distant from the ligand-binding pocket and failed to induce structural changes between the homology models. discussion we took a broad comparative approach using chimeric gal4-lbd constructs in a single assay to examine the ligand specificity of the lbds of the full range of corticosteroid receptors within single species as well as across deeply diverged vertebrate taxa. as discussed below, this property likely led to neo-functionalization and maintenance of two grs. comparisons of the amino acid sequences and predicted structures of the duplicate gr lbds added insight into the evolutionary changes that may regulate ligand specificity. in particular, the mineralocorticoid sensitivities of duplicate grs in teleosts were highly labile in the context of their molecular phylogeny, i.e., it could not be predicted based on the degree of similarity between their sequences. ligand specificity aldosterone is the primary mineralocorticoid in tetrapods, while teleost fish appear to lack the aldosterone synthase necessary to produce aldosterone. we demonstrated that teleost mr lbd is sensitive to both mineralocorticoids and glucocorticoids, consistent with previous results for both the lbd and the whole protein. the maximal activation by aldosterone was greater than that of either doc or cortisol, though the variation in ec values of these ligands for the midshipman mr construct were not substantial. doc, the potential natural agonist for mr in teleosts, elicited a response from midshipman mr. this was consistent with results for the full-length mr of rainbow trout, the mr lbd of skate, and the corticosteroid receptors of the more basal hagfish and lamprey. our data thus support the hypothesis that the evolution of an mr predates aldosterone. doc, which elicits activity from mrs across a range of vertebrates, may be the primary ligand of the ancestral mr. since doc activates the corticosteroid receptors of jawless vertebrates, the proposed ancestral corticosteroid receptor, the maintained doc sensitivity of mr may represent a conserved ancestral function. aldosterone induced greater maximal activation than doc for both mouse and midshipman mr lbd constructs with lower ec50s, suggesting that aldosterone evolved as a more potent mr agonist among lobe-finned fishes, as evidenced in extant lungfish and descendant tetrapods. the response of the mouse mr to doc, together with evidence for doc in the plasma of mammals, suggests that doc may still be an important ligand of the tetrapod mr. the mr lbds studied here showed sensitivity to both aldosterone and cortisol, as well as two steroids involved in the biosynthesis of aldosterone and cortisol. this reflects both the overall promiscuity of the mr and conserved sensitivity of the ancestral corticosteroid receptor. three of these steroids are present in the plasma of teleosts; thus, teleost mr has either a wide array of endogenous ligands or alternate mechanisms inhibit certain steroids from binding to mr, as occurs in mammals. neo-functionalization of duplicate grs the relative ligand sensitivities of gr and gr in teleosts provide insight into the biological basis for maintaining duplicate grs. bury and sturm hypothesized that the difference in sensitivity between rainbow trout grs evolved such that gr is active at basal cortisol levels while gr is recruited under stressful conditions with elevated cortisol. the gr lbds examined in this study were all activated by cortisol and with a slightly lower ec for midshipman gr than gr, though not on the order of the difference observed in rainbow trout. the dose-response data showed activity from gr at very low cortisol concentrations, while gr was not activated at these concentrations. this is further consistent with the hypothesis of gr activity restricted to times of high cortisol release. bury and sturm described the observed difference in cortisol sensitivity between duplicate grs as a neo-functionalization and proposed that the functions of a single gr are partitioned between the duplicate grs into stressful and basal-level activity. such partitioning seems more accurately described as sub-functionalization since neither of the duplicate grs acquire a novel function. the evidence presented here supports neo-functionalization based on the promiscuity of ligand specificity among gr paralogues and suggests that promiscuous grs acquired a novel function distinct from their direct ancestral predecessor. as we report for midshipman and daffodil cichlid, it appears that in many teleost species one gr shows strong ligand specificity for glucocorticoids, while the other gr responds to glucocorticoids as well as either aldosterone and doc or 11-deoxycortisol. the broader ligand sensitivity is more similar to that of the single skate gr as well as the basal hagfish and lamprey corticosteroid receptors. however, because the inferred ancestral gr of bony vertebrates is cortisol-specific, the observed promiscuousness among grs represents a novel property and thus is a neo-functionalization, rather than sub-functionalization of the ancestral characters. a similar reversion of gr to a more broadly sensitive receptor is seen in chickens in which the full-length gr is more sensitive to aldosterone than to cortisol. a promiscuous gr among bony vertebrates could function in a role similar to the ancestral gr of cartilaginous fishes, while a cortisol-specific gr serves a more selective function. the fact that gr steroid-specificity is not predictable based on the phylogeny of receptor sequences suggests that activation by steroids other than cortisol is evolutionarily labile, providing a basis for rapid adaptation of these receptors. the evolutionary plasticity of gr specificity may occasionally lead to the duplicate teleost grs reverting to redundant states and thus one of the receptors being lost in some lineages, which may be responsible for the single gr of zebrafish. amino acid substitutions and gr ligand specificity we aligned the amino acid sequences of lbds from gr and gr of midshipman with gr from burton's cichlid and gr from rainbow trout to examine the molecular basis of variation in the mineralocorticoid sensitivity of grs. nine amino acid positions correlated with mineralocorticoid sensitivity. these positions showed the same correlation with mineralocorticoid sensitivity in the daffodil cichlid gr lbds, strongly supporting a role for these residues in altering ligand specificity. five of the identified residues are noteworthy because the same amino acids occur at these positions in the mineralocorticoid-sensitive chicken gr, with residues h and f common in many mrs. furthermore, residue f has previously been shown to directly interact with the ligand, is adjacent to another ligand contacting residue, and is important for proper receptor co-activator and co-repressor binding. using homology model structures to midshipman gr and gr we identified two of the nine amino acid residues as particularly strong candidates for regulating ligand specificity because they likely either make contact with the ligand or change the structure of the lbd. the a/s substitution at position is distant from the ligand-binding pocket, but it alters the length and polarity of the distal end of helix which is likely to affect the conformation and flexibility of helix and the loop between helices and such conformational effects can play an important role in the ligand specificity of corticosteroid receptors. additionally, the f/i substitution at position is located in the binding pocket, within 4Å of the putative ligand position. though this is a substitution between two non-polar residues, this substitution introduces a change in the degree of hydrophobicity and a large difference in structure. such amino acid substitutions within the lbd affect the binding specificity and transactivational response of steroid receptors to particular ligands. our results are consistent with the findings of bridgham et al that accumulation of restrictive mutations make a reversal of protein structure and function unlikely. none of the nine candidate amino acid residues identified in our analysis correspond to residues previously suggested to play a role in the evolution of gr cortisol specificity. this incongruity indicated that mineralocorticoid sensitivity among teleost grs likely results from novel mutations that permit aldosterone binding to otherwise cortisol specific receptors rather than back-mutations to the ancestral aldosterone sensitive state.  a luciferase reporter assay was used to characterize the transcriptional activation of corticosteroid receptors across the two most widely divergent groups of bony vertebrates, tetrapods and teleosts, showing the activation of all teleost receptors by cortisol with variable activation by other steroids in the corticosteroid biosynthetic pathways. phylogenetic analyses demonstrated that gr aldosterone-sensitivity cannot be predicted among fishes on the basis of receptor sequence similarity. this suggests that different lines of teleosts have adapted duplicate grs for divergent functions. while one has cortisol-specificity, the other is broadly sensitive to corticosteroids, more akin to the ancestral state. however, the latter is not a reversal of phenotype, but instead represents a novel condition brought about by specific amino acid substitutions. such neo-functionalization reflects the dynamic and complex regulation inherent in the divergence of corticosteroid receptors across vertebrates. 
both teleost species had a single mr and duplicate grs. we used up to seven steroids to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal and the midshipman fish, a teleost model for steroid regulation of neural and behavioral plasticity. variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of mr and grs from the distantly related daffodil cichlid, another teleost model for neurobehavioral plasticity. the mineralocorticoid sensitivity of duplicate grs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two grs. teleost fishes have a single mr and duplicate grs that show variable sensitivities to mineralocorticoids and glucocorticoids. all mrs were sensitive to doc, consistent with the hypothesis that doc was the initial ligand of the ancestral mr. variation in gr steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences. corticosteroid receptors include mineralocorticoid and glucocorticoid receptors.
 corticosteroid receptors include mineralocorticoid and glucocorticoid receptors. teleost fishes have a single mr and duplicate grs that show variable sensitivities to mineralocorticoids and glucocorticoids. how these receptors compare functionally to tetrapod mr and gr, and the evolutionary significance of maintaining two grs, remains unclear.  we used up to seven steroids to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal and the midshipman fish, a teleost model for steroid regulation of neural and behavioral plasticity. variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of mr and grs from the distantly related daffodil cichlid, another teleost model for neurobehavioral plasticity. both teleost species had a single mr and duplicate grs. all mrs were sensitive to doc, consistent with the hypothesis that doc was the initial ligand of the ancestral mr. variation in gr steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences.  the mineralocorticoid sensitivity of duplicate grs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two grs. 