Evolution of Steroid Receptor Gene Families

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Evolution of Steroid Receptor Gene Families
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Lesson Today

• Evolutionary History of Function
• Gene Duplication leading to evolutionary origins
  of novel functions
• How mutations interact to modify function

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Evolution of novelty

• Gene Duplication and Subfunctionalization
• Examples
• Receptors, Enzymes, Developmental genes, etc.
• Hox clusters
• Osmoregulatory ion uptake enzymes (ATPases)
• Cytp450s (detoxification enzymes)
• Olfactory genes
• Opsin genes
• Hemoglobin

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Gene Duplications

• Main source of novel genes

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Sources of Genetic Variation (type of mutation)

• Gene duplications, followed by differentiation
• End up with gene family different opsin genes,
  hemoglobin, ATPases, etc.

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Evolution of new functions (and genes) via gene
duplications
New function
Partitioning of function
Loss of function
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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)

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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

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Steroid hormones are lipid soluble, bind to
cytoplasmic Steroid Hormone Receptors and then
enter the nucleus, leading to transcription
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• 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
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Evolutionary History of Steroid Receptors
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Estrogen Receptors
Glucocorticoid receptor
Baker, ME. 2001. Adrenal and sex steroid receptor
evolution environmental implications. Journal
of Molecular Endocrinology 26119125
Mineralocorticoid receptor
Sex steroid response probably occurred in the
early Cambrian
Progesterone receptor
Androgen receptor
Eel ERb

• Estrogen response evolved in jawless fish or
  tunicates (early chordates)

Human ERb
Trout ERa
Xenopus ERa
the most ancient of the adrenal and sex steroid
receptors
Human ERa
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Evolution of Function
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• Bridgham et al. 2006. Science. 31297

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• 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?

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Example

• Evolution of function of the aldosterone -
  Mineralocorticoid Receptor (MR) complex
• How did this ligand-receptor relationship evolve?

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• Aldosterone is thought to be a recently derived
  hormone, and a tetrapod specific hormone
  (vertebrates with four feet), absent in more
  anciently derived species

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• 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

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• The gene duplication event leading to MR and GR
  occurred gt450 million yrs ago

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Background

• Functional assays indicate that the ancestral
  (basal) receptors are activated by very low doses
  of aldosterone, cortisol, and 11-deoxycorticostero
  ne (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)

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• How might have the aldosterone-MR partnership
  have evolved?
• If the hormone is not yet present, how could
  selection drive the receptors affinity for it?
• Conversely, without the receptor, what selection
  pressure could guide the evolution of the ligand?

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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 AncCRs ligand-binding domain (see
  Fig. 1)
• Synthesized the AncCR-LBD sequence and expressed
  it in cultured cells using a reporter assay

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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)

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• WHY would the ancient corticoid receptor respond
  to a not yet existing hormone (aldosterone)?
• And how would the specificity between MR and
  aldosterone evolve?

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• 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 11b-hydroxylase
  enzyme only the tetrapod enzyme can catalyze the
  step marked with a green asterisk. (B) MRs
  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 GRs 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.

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• 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.

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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

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Which Mutations?

• Replacement of Serine106 with Proline (S106P) and
  Leucine111 with Glutamine (L111Q) conferred a
  GR-like phenotype

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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

• When each mutation was introduced in isolation,
  it was discovered that both are required to yield
  the GR phenotype

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• 2007. Science 3171544

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But now, lets 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)

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• 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

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• Fig. 2. Mechanism for switching AncGR1s ligand
  preference from aldosterone to cortisol. (A)
  Effect of substitutions S106P and L111Q on the
  resurrected AncGR1s 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 cortisols unique C17-hydroxyl (dotted
  red line).

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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 loops position is
stabilized by a hydrogen bond between Ser106 and
the backbone carbonyl of Met103.
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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
cortisols C17-hydroxyl, stabilizing the
receptor-hormone 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
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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

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• Fig. 3. Permissive substitutions in the evolution
  of receptor specificity. (A) Effects of various
  combinations of historical substitutions on
  AncGR1s 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).

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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).

• Permissive substitutions stabilized specific
  structural elements, allowing them to tolerate
  later destabilizing mutations that conferred a
  new function

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• 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.

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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

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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 receptors
  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 exploitationrecruitment of
  an older molecule, previously constrained for a
  different role, into a new functional complex