1. 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 1

2. Action of Steroid Hormones versus Gonadotropin Hormones
Peptide Hormones Steroids
Half-life in circulation short long
Speed of action fast slow
Duration of effect short long
Location of receptor membrane inside
Post-receptor regulation high low
Signal amplification high low 3

3. Mechanism of Action of Steroid Receptors
Binding protein
Steroid R
Gene Transcription
RNA
mRNA
Protein 4

4. Cellular Localization of Steroid Receptors
Early Studies (1968):
Tissue
homogenize
Cytoplasmic
fraction
Nuclear
unoccupied
occupied
centrifuge 5

5. 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

6. Cellular Localization of Steroid Receptors
Immunocytochemical studies indicated that all steroid receptors are nuclear.
Color
reaction
Receptor
antibody

7. Cellular Localization of Steroid Receptors
Cell enucleation experiments also indicated that all steroid receptors were nuclear.
centrifugation 6

8. 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

9. 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

10. 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.

11. 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

12. Nuclear Receptors
Proteins interact with steroids and other hormones that diffuse through the cell membrane.
Form hormone-receptor complexes that function as activators by binding to enhancers  hormone response elements.
Sex hormones: estrogens and androgens; glucocorticoids, cortisol, vitamin D  Ca2+ metabolism; thyroid hormone, retinoic acid  developmental factors.

13. 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.

14. Responses to hydrophobic hormones are mediated by intracellular receptors
Lipophillic
Hormone
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
Target cell
Cytoplasm
Intracellular receptor
Translation
Nuclear envelope
Transcription
Promoter
Target gene
“Hormone response element”
Nucleus

15. 2. The glucocorticoid (nuclear) receptor is found in the cytoplasm

16. 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.

17. 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

18. Specificity of DNA binding
dimerization

20. 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 7

21. 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)

22. 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.

23. Steroid Receptor Action: Roles of Heat Shock Proteins and HREs
What is the mechanism of steroid receptor activation?
hsp90 H
hsp70
hsp59 R H H
dimerize H P? H
Gene
Transcription
HRE
target gene
5’ flanking region 8

24. 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.

25. 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 9

26. 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
HBD
transcription

27. Role of Phosphorylation in Steroid Receptor Activation
Progesterone receptor transcriptional activity is inhibited by inhibitors of protein kinase A.
Progesterone
+ PKA inhibitor
hypervariable
DBD
HBD
transcription

28. 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.

29. 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
target gene
5’ flanking region

30. Steroid Receptors bind to Hormone Response Elements (HREs) on DNA
Progesterone,
Androgen,
Glucocorticoid,
Mineralocorticoid
Consensus HREs
AGAACAnnnTGTTCT
Estrogen
AGGTCAnnnTGACCT
Thyroid hormone
Retinoids
Vitamin D
AGGTCATGACCT 10

31. Palindromic Sequences Allow Binding of Receptors as Dimers
5’ -AGAACAnnnTGTTCT- 3’ H H
NNN
A T
C G
G C
TATA
EXON 1…...
Transcription

32. 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)

33. 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

34. Evidence for HRE Specificity
Mader et al., 1989
Estrogen Receptor cDNA
Transfect Cells
Expression ER
H-E2 3
Genes with ERE= transcription
Genes with GRE= no transcription 11

35. 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
DBD HBD
WT-ER
yes no estradiol
HBD
WT-GR
no yes cortisol
HBD
no yes estradiol
ER/GR 12

36. 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 13

37. 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++ 25

38. 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 18

39. Increased Expression of
Molecular Mechanism through which Glucocorticoids inhibit Inflammatory Responses
Macrophages
Interleukin-1
Protein Kinase C
Pathway
Increased Expression of
jun, fos
AP1
collagenase
dimerization
Glucocorticoid
Receptor
(-) 19

40. 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. 20

41. 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. 21

42. Activation of Progesterone Receptor by Dopamine in Absence of Progesterone
-O’Malley
Female
Lordotic
Response
C P D D1 +
RU486 22

43. Use of Antisense Oligonucleotides to Block Gene Expression
complementary
DNA sequence
Translation
Start site
AAAAA..,
PR mRNA
mRNA degradation
block translation
Block Gene Expression 27

44. Blockade of Dopamine Activation of PR by Antisense Oligonucleotides
Female
Lordotic
Response
C P D D1 +
antisense
oligo to PR 29

45. 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.

46. Estrogen Phosphorylates CREB via the MAPK Pathway – Wade & Dorsa, 2003
E2:ER
MAPK pathway
phosphorylation of CREB
gene transcription
(CRE)

47. 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 26

48. 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

49. 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.

50. Next Time…
First Midterm Exam