intracellular hormone receptors
DESCRIPTION
Intracellular Hormone Receptors. Steroid versus Peptide Hormones Mechanism of Action of Steroid Receptors Cellular Localization and Structure of Steroid Receptors How Steroid Receptors Initiate Transcription Role of Hormone Response Elements (HREs) - PowerPoint PPT PresentationTRANSCRIPT
Intracellular Hormone Receptors
Steroid versus Peptide HormonesMechanism of Action of Steroid Receptors
Cellular Localization and Structure of Steroid Receptors
How Steroid Receptors Initiate TranscriptionRole of Hormone Response Elements (HREs)Interactions of Steroid Receptors with Other
PathwaysRegulation of Steroid Receptors
Action of Steroid Hormones versusGonadotropin Hormones
Peptide Hormones Steroids
Half-life in circulation short longSpeed of action fast slowDuration of effect short longLocation of receptor membrane insidePost-receptor regulation high lowSignal amplification high low
Mechanism of Action of Steroid Receptors
Binding protein
Steroid
R
Gene Transcription
RNA
mRNA
Protein
Cellular Localization of Steroid Receptors
Early Studies (1968):
Tissue
homogenize
Cytoplasmicfraction
Nuclearfraction
unoccupied occupied
centrifuge
Localization of Protein by Immunocytochemistry
Incubate cell specimen with antibody which specifically detects the protein.
Wash away unbound antibody.
Use a second labeled antibody to detect the first antibody.
protein
Cellular Localization of Steroid Receptors
Immunocytochemical studies indicated that all steroid receptors are nuclear.
Receptorantibody
Colorreaction
Cellular Localization of Steroid Receptors
Cell enucleation experiments also indicated that all steroid receptors were nuclear.
centrifugation
Cellular Localization of Steroid Receptors
It was thought that occupied receptor had higher affinity for the nucleus than unoccupied receptor, and was thus less likely to leak out during homogenization step in earlier studies.
homogenize
Cellular Localization of Steroid Receptors
More recent immunocytochemistry experiments reveal cytoplasmic estrogen receptors in certain regions of the hypothalamus under certain conditions.
Low ER Expression Increased ER Expression
Cellular Localization of Steroid Receptors
It is generally thought that unoccupied steroid receptors can exist in the cytoplasm, while occupied receptors act in the nucleus on target DNA.
When bound to hormone, cytoplasmic receptors move into the nucleus.
Steroid Receptor Superfamily
Functionally Related: all are intracellular receptors which act as transcription factors, regulating target gene expression.
The Superfamily includes:glucocorticoid receptor
mineralocorticoid receptorprogesterone receptor (A and B)
androgen receptorestrogen receptor (alpha and beta)
thyroid hormone receptorvitamin D receptor
retinoic acid receptor
Nuclear Receptors
1. Proteins interact with steroids and other hormones that diffuse through the cell membrane.
2. Form hormone-receptor complexes that function as activators by binding to enhancers hormone response elements.
3. Sex hormones: estrogens and androgens; glucocorticoids, cortisol, vitamin D Ca2+ metabolism; thyroid hormone, retinoic acid developmental factors.
1. The majority of nuclear receptors bind to respective enhancer elements and repress transcription. - In the presence of hormone, they form R-H complexes in the nucleus and function as activators by binding to the same enhancers.- Act as repressor or enhancer, depending on the physiological signals.- thus, the response element serves as either enhancer or silencer.
Responses to hydrophobic hormones are mediated by intracellular receptors
Transcription
Translation
Cytoplasm
Nucleus
Nuclear envelope
Plasma membrane Lipophilic hormone carried in
blood
Hormone binds intracellular receptor inducing receptor dimerization and activation
Complex is imported into nucleus
Binds to “hormone response element” to regulate gene expression
Intracellular receptor
Promoter Target gene“Hormone response element”
Target cell
LipophillicHormone
2. The glucocorticoid (nuclear) receptor is found in the cytoplasm
Glucocorticoid Action
1. GR exists in an inactive form in the cytoplasm complexed with heat shock protein 90 (hsp90).
2. Glucocorticoid (G) diffuses across cell membrane and enters cytoplasm
3. G binds to GR changes conformation dissociates from hsp90
4. exposes a nuclear localization signal (stretch of aas) on GR.
5. G-GR (hormone-receptor complex, HR) enters nucleus, dimerizes with another HR.
6. HR dimer binds to enhancer/hormone-response element upstream of hormone activated gene.
7. Binding of HR dimer to enhancer activates transcription.
8. Most contain 2 zinc fingers (1) controls DNA binding, (2) controls dimerization
Critical residues for discriminating between GRE and ERE lie at the base of the first finger
-GRE = glucocorticoid responsive element /enhancer (sequence); ERE = estrogen
Specificity of DNA binding dimerization
Steroid Receptor StructureThis superfamily of ligand-activated transcription factors is also structurally related.Three well conserved regions:-Hormone binding domains (HBD) in carboxyl terminus
-DNA-binding domain (DBD) 5’ to ligand binding domainA nonconserved hypervariable region, which may contribute to transcriptional activity of receptor
DBD HBDhypervariable
How Are Steroid Hormone Receptors Activated?
We know that when bound to hormone, the hormone-receptor complex initiates transcription of target genes. But how?
- Role of Heat Shock Proteins (HSPs)
- Role of Hormone Response Elements (HREs)
Role of HSPs
Unbound receptor is associated with several HSPs.
Binding of hormone to receptor results in loss of HSPs, followed by dimerization and activation of transcription.
Steroid Receptor Action:Roles of Heat Shock Proteins and HREs
What is the mechanism of steroid receptor activation?
H
H H
dimerize
H HGene
Transcription
P?
HRE target gene
5’ flanking region
hsp90H
hsp70
hsp59
Rhsp70H
Not All Intracellular Receptors are Associated with HSPs.
HSPs bind to glucocorticoid, mineralocorticoid, androgen, progesterone, and estrogen receptors in absence of hormone.
However, receptors for thyroid hormone, retinoic acid, and vitamin D are not bound to HSPs.
This second group of receptors is bound to their hormone response element (HRE) on 5’flanking region of target genes, but are inactive until hormone binds to them.
Is Removal of HSPs Sufficient to Induce Activation of Steroid Hormone
Receptors?Removal of HSPs is not sufficient to induce transcriptional response - requires ligand.
Steroid receptors with HSP bound can still bind DNA.
Activation of steroid receptors may require a ligand-induced phosphorylation step.
Ligand-independent activation of receptors may occur if they are phosphorylated
Role of Phosphorylation in Steroid Receptor Activation
The transcriptional activity of the progesterone receptor can be stimulated by treatment with cyclic AMP.
DBD HBDhypervariable
8-bromo cyclic AMP
PKA?
+ Phosphate
transcription
• Progesterone receptor transcriptional activity is inhibited by inhibitors of protein kinase A.
DBD HBDhypervariable
Progesterone
transcription
+ PKA inhibitor
Role of Phosphorylation in Steroid Receptor Activation
• Occupancy of steroid receptors by hormone is associated with increased phosphorylation of the receptor.
• Thus, phosphorylation of steroid receptors appears to be an important step in receptor activation.
Role of Phosphorylation in Steroid Receptor Activation
Following hormone binding, intracellular receptors act as transcription factors, binding to hormone response elements (HREs) on the 5’ flanking region of target genes.
HRE target gene
5’ flanking region
Steroid Receptors bind to Hormone Response Elements (HREs) on DNA
Steroid Receptors bind to Hormone Response Elements (HREs) on DNA
HormoneProgesterone,Androgen,Glucocorticoid,Mineralocorticoid
Consensus HREsAGAACAnnnTGTTCT
Estrogen
Thyroid hormoneRetinoidsVitamin D
AGGTCAnnnTGACCT
AGGTCATGACCT
Palindromic Sequences Allow Binding of Receptors as Dimers
5’ -AGAACAnnnTGTTCT- 3’
H HNNNA TC GA TA TG C A T
TATA EXON 1…...
Transcription
Sharing of HREs by Different Steroid Receptors
Note that there are 8 classes of steroid receptors, but only 3 consensus HRE’s. Many receptors recognize the same HRE!How is specificity achieved (how does a cell know its being stimulated by PR and not GR)?
- Cell specific expression of receptors (don’t express both PR and GR in same cell. But sometimes they are in the same cell!)
- Other transcriptional regulation elements (cofactors)
- Formation of heterodimers versus homodimers (ie, thyroid hormone receptor with retinoic acid receptor)
Role of Cofactors in Steroid Receptor Action and Specificity
There are cofactors that interact with steroid receptors to facilitate increased transcription
Example: Cdc37 interacts with the androgen receptor, plays a role in transcriptional response.
Cdc37 affects protein folding.
Cdc37 does not interact with glucocorticoid receptor (which shares the same HRE on target DNA)
---AGAACAnnnTGTTCT--target gene ARE/GRE
Androgen Receptor Glucocorticoid Receptor
Cdc37
Evidence for HRE Specificity
Mader et al., 1989
Estrogen Receptor cDNA
Transfect Cells
ERH-E23
Expression
Genes with ERE= transcriptionGenes with GRE= no transcription
Specificity of HRE/DNA Binding Domain Interaction
DBD HBD ERE binding GRE binding Ligand
WT-ER
HBD
WT-GR
HBD
ER/GR
yes no estradiol
no yes cortisol
no yes estradiol
How do we know it’s the HRE and DNA Binding Domain that interact to give specificity of transcriptional regulation? Here’s an experiment.
How does binding of Activated Steroid Receptor to HREs Enhance Transcription?HREs are enhancer sequences: they are orientation- and distance-independent
Binding of activated receptor to HRE may stabilize the interactions between TATA box, Transcription Factor IID, and RNA polymerase II
intron
exon
ERE TATA BOX
5’-flanking region
Do steroids always act through classical steroid receptor mechanisms?
Some effects of steroids are observed in minutes, too quickly to be explained by regulation of transcription.
Rapid effects of steroids may involve changes in ion channels and membrane permeability, such as influencing membrane potentials in CNS neurons.
In human sperm, a membrane-bound progesterone “receptor” has been described, which may mediate the effects of P on sperm maturation.
progesteroneCa++
Interactions of Steroid Receptors with Regulatory Elements of other Signaling
Pathways
• I previously mentioned that the transcriptional activity of the chick PR is increased by cAMP in the absence of progesterone. Protein kinase A inhibitors block this effect.
We also know that:• Glucocorticoid receptors interact with the transcription factor AP1• Progesterone receptors can be activated by dopamine (a neurotransmitter)• Thyroid hormone receptor activity is inhibited by AP1
Molecular Mechanism through which Glucocorticoids inhibit Inflammatory
Responses
Macrophages Interleukin-1
Protein Kinase CPathway
Increased Expression ofjun, fos
AP1 collagenase
dimerizationGlucocorticoid
Receptor
(-)
Inhibition of AP-1 by GR
Glucocorticoid receptor may bind jun, decreasing the formation of AP-1 (jun/fos dimer). This results in less AP-1 to bind to the the AP-1 enhancer site on the 5’ flanking region of the collagenase gene.
However, DNA footprinting studies show that AP-1 still DOES bind the AP-1 site during GR-induced inhibition of AP-1 stimulated transcription.
GR may inhibit transcriptional activation by AP-1 once bound to the site.
Activation of Progesterone Receptor by Dopamine in absence of Progesterone
Dopamine is a common neurotransmitter in the brain.
Dopamine D1 receptor agonists mimic the effects of progesterone on female mating behavior.
The effects of dopamine can be blocked by a progesterone receptor antagonist and by antisense oligonucleotides to the progesterone receptor.
Activation of Progesterone Receptor by Dopamine in Absence of Progesterone
-O’Malley
FemaleLordoticResponse
C P D1 D1 +
RU486
Use of Antisense Oligonucleotides to Block Gene Expression
TranslationStart site
AAAAA..,PR mRNA
complementaryDNA sequence
Block Gene Expression
mRNA degradationblock translation
Blockade of Dopamine Activation of PR by Antisense Oligonucleotides
FemaleLordoticResponse
C P D1 D1 + antisense
oligo to PR
Estrogen Phosphorylates CREB via the MAPK Pathway – Wade & Dorsa, 2003
Treatment of brain cells with estrogen results in rapid phosphorylation (15 min) of CREB.This effect is blocked by an estrogen receptor antagonist (ER dependent)This effect is dependent upon the activity of the mitogen-activated protein kinase (MAPK) pathway.This effect is NOT dependent upon activity of protein kinase A.
Estrogen Phosphorylates CREB via the MAPK Pathway – Wade & Dorsa, 2003
E2 E2:ER
MAPK pathway
phosphorylation of CREB gene transcription(CRE)
Regulation of Steroid Hormone Receptor Expression
In general, tissue-specific and hormone-mediated regulation of steroid hormone receptors is not as dramatic as that for peptide hormone receptors.Autoregulation: Ligand influences expression of own receptor.Autoregulation can occur at several levels:
- transcriptional: control of gene expression- post-transcriptional: modulation of mRNA stability- post-translational: rate of receptor degradation
Examples:-Estradiol decreases uterine ER expression-Estradiol increases ER in pituitary, liver
Regulation of Steroid Hormone Receptor Expression
Heterologous regulation - Regulation by other steroids:
-Estrogen up-regulates progesterone receptor in breast, uterus, hypothalamus
-Progesterone down-regulates estrogen receptor
-Androgen down-regulates estrogen receptor
Influences of Other Signaling Pathways
Steroid receptor expression may also be influenced by the protein kinase pathways.
Example: Activation of PKC decreases estrogen receptor mRNA stability, resulting in decreased synthesis.
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First Midterm Exam