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)
Interactions of Steroid Receptors with Other
Pathways
Regulation of Steroid Receptors
Action of Steroid Hormones versus
Gonadotropin Hormones
Peptide Hormones
Half-life in circulation
Speed of action
Duration of effect
Location of receptor
Post-receptor regulation
Signal amplification
short
fast
short
membrane
high
high
Steroids
long
slow
long
inside
low
low
Mechanism of Action of Steroid
Receptors
Gene Transcription
RNA
mRNA
R
Protein
Steroid
Binding protein
Cellular Localization of Steroid
Receptors
Early Studies (1968):
Tissue
homogenize
centrifuge
Cytoplasmic
fraction
Nuclear
fraction
unoccupied
occupied
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.
Receptor
antibody
Color
reaction
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 receptor
progesterone receptor (A and B)
androgen receptor
estrogen receptor (alpha and beta)
thyroid hormone receptor
vitamin D receptor
retinoic acid receptor
Nuclear Receptors
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.
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
Lipophillic
Hormone
Plasma
membrane
Target
cell
Cytoplasm
Intracellular
receptor
Nuclear
envelope
Transcription
Promoter
Hormone binds intracellular
receptor inducing receptor
dimerization and activation
Complex is imported into nucleus
Translation
“Hormone
response
element”
Lipophilic hormone carried in
blood
Target gene
Nucleus
Binds to “hormone response
element” to regulate gene
expression
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 Structure
This 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 domain
A nonconserved hypervariable region, which
may contribute to transcriptional activity of
receptor
hypervariable
DBD
HBD
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?
hsp90
hsp70
H
H
hsp70
H
R
hsp59
dimerize
H
P?
Gene
Transcription
H
H
HRE
5’ flanking region
target gene
H
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.
8-bromo cyclic AMP
PKA?
+ Phosphate
hypervariable
DBD
transcription
HBD
Role of Phosphorylation in Steroid
Receptor Activation
• Progesterone receptor transcriptional activity
is inhibited by inhibitors of protein kinase A.
Progesterone
+ PKA inhibitor
hypervariable
DBD
transcription
HBD
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.
Steroid Receptors bind to Hormone
Response Elements (HREs) on DNA
Following hormone binding, intracellular
receptors act as transcription factors,
binding to hormone response elements
(HREs) on the 5’ flanking region of target
genes.
HRE
5’ flanking region
target gene
Steroid Receptors bind to Hormone
Response Elements (HREs) on DNA
Hormone
Progesterone,
Androgen,
Glucocorticoid,
Mineralocorticoid
Consensus HREs
AGAACAnnnTGTTCT
Estrogen
AGGTCAnnnTGACCT
Thyroid hormone
Retinoids
Vitamin D
AGGTCATGACCT
Palindromic Sequences Allow
Binding of Receptors as Dimers
5’ -AGAACAnnnTGTTCT- 3’
H
H
NNN
A T
C G
A T
A T
G C
A T
Transcription
TATA
EXON 1…...
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)
Cdc37
Androgen Receptor
Glucocorticoid Receptor
---AGAACAnnnTGTTCT--target gene
ARE/GRE
Evidence for HRE Specificity
Mader et al., 1989
Estrogen Receptor cDNA
Transfect Cells
Expression
3
H-E2 ER
Genes with ERE= transcription
Genes with GRE= no transcription
Specificity of HRE/DNA Binding
Domain Interaction
How do we know it’s the HRE and DNA Binding
Domain that interact to give specificity of transcriptional
regulation? Here’s an experiment.
ERE binding
GRE binding Ligand
WT-ER
yes
no
estradiol
WT-GR
no
yes
cortisol
ER/GR
no
yes
estradiol
DBD HBD
HBD
HBD
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
5’-flanking region
intron
exon
ERE TATA
BOX
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.
progesterone
Ca++
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 C
Pathway
Increased Expression of
jun, fos
(-)
Glucocorticoid
Receptor
dimerization
AP1
collagenase
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
Female
Lordotic
Response
C
P
D1
D1
+
RU486
Use of Antisense Oligonucleotides to
Block Gene Expression
complementary
DNA sequence
AAAAA..,
PR mRNA
Translation
Start site
mRNA degradation
block translation
Block Gene Expression
Blockade of Dopamine Activation of
PR by Antisense Oligonucleotides
Female
Lordotic
Response
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.
Next Time…
First Midterm Exam