A central goal in studies of molecular evolution is to reveal the genetic, structural, and biophysical mechanisms by which protein functions have evolved 
. Ancient proteins and DNA are seldom directly available, but the traces of their evolutionary history are found in their extant descendants 
. Direct comparisons among present-day proteins can sometime yield insights into the sequence and structural mechanisms that underlie functional differences 
. Such “horizontal” comparisons, however, cannot determine which protein features are ancestral and which are derived, so they are not suited to reconstructing the events that produced functional diversity 
. Further, because the effect of a mutation on protein structure and function often depends on the residues present at other sequence sites 
, studies of extant proteins may often be unsuited to revealing the effects of mutations in the historical backgrounds in which they occurred 
Ancestral sequence reconstruction (ASR) allows the forms and functions of ancient proteins to be studied experimentally. Beginning with an alignment of extant sequences, the maximum likelihood phylogeny and best-fit probabilistic model of evolution are inferred; the most likely ancestral sequence at any node – defined as the sequence with the highest probability of delivering all the observed extant sequences – can then be identified 
. These ancestral protein sequences can be “resurrected” using gene synthesis and cell culture or in vitro
expression systems and then characterized using the same methods typically applied to study extant proteins. This approach allows hypotheses about the ancestral and derived characteristics of proteins to be tested experimentally. It also allows the historical interval during which structure and function changed to be identified and the causal role of specific historical mutations in the ancestral background to be determined.
The glucocorticoid and mineralocorticoid receptors (GR and MR) are paralogous hormone-regulated transcription factors that have served as useful models for studying protein evolution 
. GR and MR have a modular domain structure that includes a well-conserved DNA-binding domain (DBD) and a moderately conserved ligand-binding domain (LBD) – which binds the hormone, changes conformation, and attracts coactivator proteins that potentiate transcription of nearby target genes; they also contain poorly conserved hinge and N-terminal domains. In most bony vertebrates, the intrinsic functions of the GR and MR LBDs differ in both specificity and sensitivity. GR is more specific, being activated by high doses of the adrenal hormone cortisol to regulate aspects of immunity, glucose metabolism, and the long-term stress response 
. MR, in contrast, is activated by the adrenal mineralocorticoids aldosterone or deoxycorticosterone, as well as cortisol (albeit with somewhat lower sensitivity), and primarily regulates osmotic homeostasis. GR is also considerably less sensitive than MR, often requiring concentrations several orders of magnitude higher for activation 
Some information is available on GR and MR evolution. The two paralogs descend by duplication from a single ancestral corticosteroid receptor (AncCR), which existed in an ancient jawed vertebrate ~450 million years ago, before the divergence of bony vertebrates from cartilaginous fishes () 
. Reconstruction and experimental analysis showed that AncCR, like the extant MRs, was extremely sensitive to both mineralocorticoids and glucocorticoids, and its structure was MR-like, as well 
. Subsequent work revealed that GR's specificity for glucocorticoids evolved later in the lineage leading to bony vertebrates, after the divergence of cartilaginous fishes but before the split of ray-finned fish from the lineage leading to tetrapods and lobe-finned fish, due to a small specific set of historical mutations 
Simplified phylogeny of corticosteroid receptors.
The evolutionary causes of GR's reduced hormone sensitivity are not known. In the little skate – the only cartilaginous fish studied to date – GR is a low-sensitivity, broad-spectrum receptor: like MR, it responds to both glucocorticoids and mineralocorticoids, but it is unique in requiring high concentrations of either type of hormone to activate it. The difference in receptor sensitivity between the GR and MR is thought to have physiological consequences: in several elasmobranch species, the same corticosteroids appear to regulate both stress and osmolarity 
, and the highest titres are associated with stress conditions 
. These observations suggest that GR regulates stress in response to high doses of hormones, while MR regulates osmolarity in response to much lower doses 
Based on these data, we hypothesize that GRs' reduced sensitivity to all hormones was an independent evolutionary event that occurred before cartilaginous fishes split from bony vertebrates, and before glucocorticoid specificity evolved in the GRs of bony vertebrates 
. Here we report on experiments to test this hypothesis and determine the genetic, structural, and biophysical mechanisms by which GR's reduced hormone sensitivity evolved. We first resurrected the LBD of AncGR1 () – the GR protein present in the common ancestor of bony and cartilaginous vertebrates and the earliest node after the GR-MR split – and then used functional assays, X-ray crystallography, site-directed mutagenesis, and computational predictions of biophysical parameters to dissect the mechanisms by which GR evolved. We show that after its initial birth by gene duplication, a small number of mutations that partially degraded its structure, stability, and function caused GR to become a novel low-sensitivity receptor.