Other Mechanisms of Nuclear Receptor Activation

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Other Mechanisms of Nuclear Receptor Activation

• Ligand Independent Activation
• Steve Sizemore

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Ligand Independent Activation Crosstalk

• Nuclear Receptor (NR) Family members are
  classically viewed as ligand activated
  Transcription Factors.
• This view has been expanded as it has became
  apparent that the stimulation of some kinase
  pathways can lead to the activation of some NR
  family members in the absence of their cognitive
  ligand (ligand independent activation)
• These kinases are regulated by a variety of
  signaling pathways stimulated by growth factors,
  cytokines and neurotransmitters.

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Cross-talk between SRs and other signaling
pathways (httppicard.ch/DP/Factors.pdf
)Pathways known to induce steroid-independent
activation of Steroid Receptors
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Ligand-Independent Activation

• Relies upon phosphorylation
• Most phosphorylation sites lie in the variable
  ,N-terminal, AF-1 domain.
• A serine-proline motif is common to many NR
  phosphorylation sites

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Ligand-Independent Activation

• Trans-activation by AF-1 depends upon interaction
  of phospho-residues and co-activators.

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Example of Ligand-Independent Activation

• EGF activation of the ER is the best
  characterized example
• EGF treatment can mimic the effect of estrogen in
  the uterus.
• This is not observed in ER knockout mice
• Agents that increase cAMP and stimulate PKA can
  also mimic the effect of estrogen

• Activation of the ER by EGF is mediated by the
  MAP
• kinase pathway and leads to phosphorylation
  of Ser 118
• of the AF-1 domain.

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Points to Remember

• Growth factors and even binding proteins (SHBG)
  found in cell culture media can influence the
  activity of some nuclear receptors via
  ligand-independent activation mechanisms.
• Transformed cell-lines often have deviant signal
  transduction pathways which may result in
  unexpected ligand-independent activation of
  Nuclear Receptor family members.

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Ligand-Independent Activation and Antagonists

• Many competitive inhibitors of SHRs are mixed
  agonists/antagonists (Tamoxifen is an example of
  a mixed agonist/antagonist of the ER)
• These bind the ligand binding site, allow
  receptor dimerization, translocation and DNA
  binding but prevent AF-2 activation.

• Mixed agonists/antagonists do not inhibit
  trans-activation through
• AF-1! (and may actually cause AF-1
  activation)
• In tissues where AF-2 function is required, a
  mixed
• antagonist/agonist will act as an antagonist
  (e.g. Tamoxifen
• in breast tissue) .
• In tissues where the AF-1 domain is active, a
  mixed
• agonist/antagonist may act as an agonist (e.g.
  Tamoxifen in
• bone).

• Pure antagonists like ICI182,780 (another
  anti-estrogen) block
• trans-activation through both AF-2 and AF-1.

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Ligand-Independent Activation and Antagonists
Ligand-independent activation of the ER is not
blocked by the mixed estrogen agonist/antagonist
Tamoxifen.
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Points to Remember

• Mixed agonist/antagonists like Tamoxifen may not
  inhibit activation of Nuclear Receptors via
  ligand-independent mechanisms.
• True antagonists, like ICI182,780, will inhibit
  both ligand-dependent and ligand-independent
  activation of nuclear receptors.

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Ligand-Independent Activation of AF-2?

• Ligand independent activation of the AF-2
  trans-activation domain may also be possible.
• ER has a Tyrosine residue at position 537 in the
  LBD.
• Site directed mutants in which this tyrosine is
  replaced by aspartic acid, glutamic acid, or
  alanine show constitutive ER activity.
• It is believed that Tyr 537 is important for
  hydrophobic interactions that maintain the
  receptor in an inactive state.
• Replacing this Tyr with a more hydrophilic
  residue disrupts these interactions and favors
  the active conformation.
• Conversion of the tyrosine at 537 to a more
  hydrophilic phospho-tryosine may also allow for
  AF-2 activation in absence of ligand.
• This tyrosine residue is highly conserved in NR
  family members (see residue 530 in the chicken PR
  schematic below).
• The Auricchio lab has noted that this residue is
  phosphorylated in vivo.
• Currently, there is no known kinase pathway that
  phosphorylates this residue.

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Closing Comments

• Note that not all phosphorylations of NR family
  members will result in ligand-independent
  activation
• Ligand-independent activation is cell and
  promoter specific.
• Some changes in phosphorylation state are due to
  ligand binding.
• Other changes in phosphorylation state may
  influence ligand binding, dimerization or DNA
  binding.
• Ligand-dependent activation of NRs and ligand-
  independent activation are not mutually exclusive
  and often may be synergistic.

• The mechanisms of ligand-independent activation
  are best understood
• and characterized in the Steroid Receptors
  but the other members of
• the NR family are also phosphoproteins and
  may be activated by
• ligand-independent means. Currently, there
  is evidence for ligand-
• independent activation of TR, RXR, RAR, and
  VDR in addition to
• the SRs.