Transcript Evolution of Steroid Receptors

Evolution of Steroid Receptor
Gene Families
Lesson Today
• Evolutionary History of Function
• Gene Duplication leading to evolutionary
origins of novel functions
• How mutations interact to modify function
Evolution of novelty
Gene Duplication and Subfunctionalization:
Examples:
Receptors, Enzymes, Developmental genes,
etc.:
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Hox clusters
Osmoregulatory ion uptake enzymes (ATPases)
Cytp450s (detoxification enzymes)
Olfactory genes
Opsin genes
Hemoglobin
Gene Duplications
• Main source of novel genes
Sources of Genetic Variation
(type of mutation)
• Gene duplications,
followed by differentiation
• End up with “gene family”:
different opsin genes,
hemoglobin, ATPases,
etc.
Evolution of new functions (and genes) via gene duplications
Loss of function
New function
Partitioning of function
What are Steroid Receptors?
I am using this as an example to facilitate
understanding of the impacts of pesticides
and other environmental toxins on animal
physiology (next lectures)
Steroid Hormone Receptors
• Transcription factors
• Intracellular receptors (typically cytoplasmic)
that bind to ligands (e.g. steroid hormones)
• Initiate signal transduction which lead to
changes in gene expression
Steroid hormones are lipid soluble, bind to cytoplasmic
Steroid Hormone Receptors and then enter the nucleus,
leading to transcription
The estrogen receptor is
fairly nonspecific,
• It is ancestral (phylogeny on
next slide), and ancestral
receptors tend to be less
specific (specificity evolves)
• It needs to bind to multiple
ligands, ~12 estrogens
(estradiol, estriol, estrone, etc.)
So... many compounds will
bind to it, such as pesticides
Estrogen receptor alpha ligand-binding
domain complexed to estradiol
Evolutionary History of Steroid Receptors
Baker, ME. 2001. Adrenal and sex steroid receptor
evolution: environmental implications. Journal of
Molecular Endocrinology 26:119–125
Glucocorticoid
receptor
Sex steroid
response
probably
occurred in the
early Cambrian
Mineralocorticoid
receptor
Progesterone
receptor
Androgen
receptor
Estrogen response
evolved in jawless fish or
tunicates (early
chordates)
Eel ERb
Human ERb
Trout ERa
Xenopus ERa
Human ERa
Estrogen
Receptors
the most ancient of
the adrenal and sex
steroid receptors
Evolution of Function
Bridgham et al. 2006. Science. 312:97
How would an integrated molecular system
evolve, such as the functional interaction between
a hormone and receptor?
For example, how could a hormone evolve if a
receptor is not present, and visa versa?
Example
• Evolution of function of the aldosterone Mineralocorticoid Receptor (MR) complex
• How did this ligand-receptor relationship
evolve?
• Aldosterone is thought to be a recently
derived hormone, and a tetrapod
specific hormone (vertebrates with four
feet), absent in more anciently derived
species
• Mineralocorticoid receptor (MR) and the
Glucocorticoid receptor (GR) descend from a gene
duplication deep in the vertebrate lineage (~450+ mya)
and now have distinct signaling functions
• In most vertebrates, GR is activated by the stress
hormone cortisol to regulate metabolism, inflammation,
and immunity
• MR is activated by aldosterone to regulate reabsorption of
ions and water and secretion of potassium in the kidneys.
MR can also be activated by cortisol
The gene duplication
event leading to MR
and GR occurred
>450 million yrs ago
Background
• Functional assays indicate that the ancestral (basal)
receptors are activated by very low doses of aldosterone,
cortisol, and 11-deoxycorticosterone (DOC); they are
similar in this respect to MRs of tetrapods and teleosts
(Fig. 2 -next slide)
• The only receptors insensitive to aldosterone are the GRs
of tetrapods and teleosts
• Given these results, the most parsimonious scenario is
that AncCR was capable of being activated by
aldosterone and that aldosterone sensitivity was lost in
the GRs of bony vertebrates (see Fig. 1)
• How might have the aldosterone-MR
partnership have evolved?
• If the hormone is not yet present, how could
selection drive the receptor’s affinity for it?
• Conversely, without the receptor, what selection
pressure could guide the evolution of the ligand?
Test Hypothesis:
• Performed gene resurrection to experimentally
examine the function of the ancestral corticoid
receptor (AncCR)
• Inferred the maximum likelihood (ML) amino acid
sequence of AncCR’s ligand-binding domain (see
Fig. 1)
• Synthesized the AncCR-LBD sequence and
expressed it in cultured cells; using a reporter assay
Results
• AncCR is a sensitive and effective aldosterone
receptor (Fig. 3A)
• Like the extant CRs and MRs, it is also activated by
low doses of DOC and, to a lesser extent, cortisol
(Fig. 3A)
• This result is surprising, because aldosterone has
long been considered a tetrapod-specific hormone
• Aldosterone is absent from the plasma of lamprey
and hagfish (more ancient vertebrates) (Fig. 3B)
• WHY would the ancient corticoid
receptor respond to a not yet existing
hormone (aldosterone)?
• And how would the specificity between
MR and aldosterone evolve?
Fig. 4. Evolution of specific aldosterone-MR signaling by molecular
exploitation. (A) Synthesis pathway for corticosteroid hormones. Ligands
for the ancestral CR and extant MRs are underlined; cortisol, the ligand for
the tetrapod GR, is overlined. The terminal addition of aldosterone is in
green. Asterisks, steps catalyzed by the cytochrome P-450 11bhydroxylase enzyme; only the tetrapod enzyme can catalyze the step
marked with a green asterisk. (B) MR’s aldosterone sensitivity preceded
the emergence of the hormone. The vertebrate ancestor did not synthesize
aldosterone (dotted circle), but it did produce other corticosteroids (filled
circle); it had a single receptor with affinity for both classes of ligand. A
gene duplication (blue) produced separate GR and MR. Two changes in
GR’s sequence (red) abolished aldosterone activation but maintained
cortisol sensitivity [see (C)]. In tetrapods, synthesis of aldosterone
emerged due to modification of cytochrome P-450 11b-hydroxylase. mya,
million years ago. (C) Mechanistic basis for loss of aldosterone sensitivity
in the GRs. Phylogenetically diagnostic amino acid changes that occurred
during GR evolution were introduced into AncCR-LBD by mutagenesis.
Dose-response is shown for aldosterone (green), DOC (blue), and cortisol
(red). The double mutant (bottom right) has a GR-like phenotype. Arrows
shows evolutionary paths via a nonfunctional (red) or functional (green)
intermediate.
Extant MRs retain the ancestral phenotype, so the
specificity of the MR-aldosterone relationship is
actually due to the secondary loss of aldosterone
sensitivity in the GR (Fig. 4B), rather than evolution
of specificity for MR.
Which Mutations?
• Explored which sequence changes are on the
branch where aldosterone sensitivity was lost
• Introduced all four single GR-diagnostic
states and all six two-fold combinations into
AncCR-LBD using mutagenesis and
determined their effect on receptor function
Which Mutations?
• Replacement of Serine106 with Proline (S106P) and
Leucine111 with Glutamine (L111Q) conferred a GRlike phenotype
When each mutation
was introduced in
isolation, it was
discovered that both
are required to yield
the GR phenotype
• L111Q alone radically reduces activation by all ligands tested
• S106P reduces aldosterone (green) and cortisol (red) sensitivity, but
this receptor remains highly DOC-sensitive (blue)
• In the S106P background, L111Q further reduces aldosterone
sensitivity but now restores cortisol response to levels characteristic of
extant GRs
2007. Science 317:1544
But now, let’s look
more closely at the
actual transition
where the mutations
occur
• These substitutions recapitulate a large
portion of the functional shift from
AncGR1 to AncGR2 (~420 to 440 Ma),
radically reducing aldosterone and DOC
response while maintaining moderate
sensitivity to cortisol (Fig. 2A)
• Instead of using the ancestral AncCR, the
structures of AncGR1 and AncGR2 were
compared to determine the mechanism by which
these two substitutions shift function
• Ancient GR1 and GR2 were reconstructed using
homology modeling and energy minimization
based on the AncCR and human GR crystal
structures
Fig. 2. Mechanism for switching AncGR1’s ligand preference from aldosterone to cortisol. (A) Effect of substitutions
S106P and L111Q on the resurrected AncGR1’s response to hormones. Dashed lines indicate sensitivity to
aldosterone (green), cortisol (purple), and DOC (orange) as the EC50 for reporter gene activation. Green arrow shows
probable pathway through a functional intermediate; red arrow, intermediate with radically reduced sensitivity to all
hormones. (B) Structural change conferring new ligand specificity. Backbones of helices 6 and 7 from AncGR1 (green)
and AncGR2 (yellow) in complex with cortisol are superimposed. Substitution S106P induces a kink in the interhelical
loop of AncGR2, repositioning sites 106 and 111 (arrows). In this background, L111Q forms a new hydrogen bond with
cortisol’s unique C17-hydroxyl (dotted red line).
The major structural difference
between AncGR1 and AncGR2
involves Helix 7 and the loop preceding
it, which contain S106P and L111Q and
form part of the ligand pocket (Fig. 2B).
In AncGR1 and AncCR, the loop’s position
is stabilized by a hydrogen bond between
Ser106 and the backbone carbonyl of
Met103.
The movement of helix 7 dramatically repositions
site 111, bringing it close to the ligand
In this conformational background,
L111Q (leucine to glutamine) generates
a hydrogen bond with cortisol’s C17hydroxyl, stabilizing the receptorhormone complex. Aldosterone and
DOC lack this hydroxyl, so the new bond
is cortisol specific
Replacing Ser106 with proline in the derived GRs
breaks this H bond and introduces a sharp kink into
the backbone, which pulls the loop downward,
repositioning and partially unwinding helix 7
The two substitutions
destabilize the receptor complex
with aldosterone or DOC
Achieves stability with cortisol,
switching preference to that
hormone
This mode of structural evolution is termed “conformational
epistasis” because one substitution remodels the protein
backbone and repositions a second site, changing the
functional effect of substitution at the second site
Fig. 3. Permissive substitutions in the evolution of receptor specificity. (A) Effects of various combinations of historical substitutions on AncGR1’s transcriptional
activity and hormonesensitivity in a reporter gene assay. Group Y (L29M, F98I, and S212D) abolishes receptor activity unless groups X (S106P, L111Q) and Z
(N26T and Q105L) are present; the XYZ combination yields complete cortisol-specificity. The 95% confidence interval for each EC50 is in parentheses. Dash, no
activation. (B) Structural prediction of permissive substitutions. Models of AncGR1 (green) and AncGR2 (yellow) are shown with cortisol. Group X and Y
substitutions (circles and rectangles) yield new interactions with the C17-hydroxyl of cortisol (purple) but destabilize receptor regions required for activation. Group
Z (underlined) imparts additional stability to the destabilized regions. (C) Restricted evolutionary paths through sequence space. The corners of the cube represent
states for residue sets X, Y, and Z. Edges represent pathways from the ancestral sequence (AncGR1) to the cortisol-specific combination (+XYZ). Filled circles at
vertices show sensitivity to aldosterone (green), DOC (orange), and cortisol (purple); empty circles, no activation. Red octagons, paths through nonfunctional
intermediates; arrows, paths through functional intermediates with no change (white) or switched ligand preference (green).
Permissive substitutions stabilized
specific structural elements,
allowing them to tolerate later
destabilizing mutations that
conferred a new function
Evolutionary trajectories that pass through functional
intermediates are more likely than those involving
nonfunctional steps, so the only historically likely pathways to
AncGR2 are those in which the permissive substitutions of
group Z and the large-effect mutations of group X occurred
before group Y was complete (Fig. 3C).
Fig. 4. Structural identification of an ancient permissive substitution. (A) Comparison of the structures of AncCR
(blue) and AncGR2 (yellow). Y27R generates a novel cation-p interaction in AncGR2 (dotted cyan line),
replacing the weaker ancestral hydrogen bond (dotted red) and imparting additional stability to helix 3. (B) Y27R
is permissive for the substitutions that confer GR function. Reporter gene activation by AncGR1 + XYZ (upper
right) is abolished when Y27R is reversed (lower right). (Left) Y27R has negligible effect in the AncCR
background (or in AncGR1, fig. S9). Green, orange, and purple lines show aldosterone, DOC, and cortisol
responses, respectively. Green arrows, likely pathway through functional intermediates.
Evolution of specificity of function
• Structural studies of human GR have shown that these
two residues change the architecture of the ligand-binding
pocket and alter contacts with steroid in ways that exclude
aldosterone and facilitate cortisol activation
• Results indicate that aldosterone specificity of MR arose
from two crucial Amino Acid replacements in the GRs that
wiped out ancestral sensitivity to aldosterone
• These changes result in evolution of a more
specific endocrine response, allowing electrolyte
homeostasis to be controlled without also
triggering the GR stress response
Molecular Exploitation
• Functional interaction between aldosterone and
mineralocorticoid receptor evolved by a stepwise selective
process
• Ancestral gene resurrection demonstrates that long before the
hormone evolved, the receptor’s affinity for aldosterone was
present due to its similarity to more ancient ligands (probably
DOC)
• Two amino acid changes in the ancestral sequence resulted in
the evolution of present-day receptor specificity
• Results indicate that tight interactions could evolve by
molecular exploitation—recruitment of an older molecule,
previously constrained for a different role, into a new
functional complex