Intracellular hormone receptors
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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)

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Intracellular Hormone Receptors

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Intracellular hormone receptors

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

Action of Steroid Hormones versusGonadotropin Hormones

Peptide HormonesSteroids

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


Mechanism of action of steroid receptors

Gene Transcription

RNA

mRNA

R

Protein

Steroid

Binding protein

Mechanism of Action of Steroid Receptors


Cellular localization of steroid receptors

Tissue

homogenize

centrifuge

Cytoplasmic

fraction

Nuclear

fraction

unoccupied

occupied

Cellular Localization of Steroid Receptors

Early Studies (1968):


Localization of protein by immunocytochemistry

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 receptors1

Receptor

antibody

Color

reaction

Cellular Localization of Steroid Receptors

  • Immunocytochemical studies indicated that all steroid receptors are nuclear.


Cellular localization of steroid receptors2

centrifugation

Cellular Localization of Steroid Receptors

  • Cell enucleation experiments also indicated that all steroid receptors were nuclear.


Cellular localization of steroid receptors3

homogenize

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.


Cellular localization of steroid receptors4

Low ER Expression

Increased ER Expression

Cellular Localization of Steroid Receptors

  • More recent immunocytochemistry experiments reveal cytoplasmic estrogen receptors in certain regions of the hypothalamus under certain conditions.


Cellular localization of steroid receptors5

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

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

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.


Intracellular hormone receptors

  • 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

Promoter

Target gene

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

“Hormone response element”

Nucleus


Intracellular hormone receptors

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


Intracellular hormone receptors

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.


Intracellular hormone receptors

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


Intracellular hormone receptors

dimerization

Specificity of DNA binding


Steroid receptor structure

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

DBD

HBD

hypervariable


How are steroid hormone receptors activated

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

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

hsp90

H

hsp70

H

H

R

hsp70

hsp59

H

H

P?

H

H

target gene

HRE

5’ flanking region

Steroid Receptor Action:Roles of Heat Shock Proteins and HREs

What is the mechanism of steroid receptor activation?

dimerize

Gene

Transcription


Not all intracellular receptors are associated with hsps

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

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

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

DBD

HBD

hypervariable

transcription


Intracellular hormone receptors

Role of Phosphorylation in Steroid Receptor Activation

  • Progesterone receptor transcriptional activity is inhibited by inhibitors of protein kinase A.

Progesterone

+ PKA inhibitor

DBD

HBD

hypervariable

transcription


Intracellular hormone receptors

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

target gene

HRE

5’ flanking region

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.


Steroid receptors bind to hormone response elements hres on dna1

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

H

H

NNN

A T

C G

A T

A T

G C

A T

Transcription

TATA

EXON 1…...

Palindromic Sequences Allow Binding of Receptors as Dimers

5’ -AGAACAnnnTGTTCT- 3’


Sharing of hres by different steroid receptors

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

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

Estrogen Receptor cDNA

Transfect Cells

Expression

3

H-E2

ER

Evidence for HRE Specificity

Mader et al., 1989

Genes with ERE= transcription

Genes with GRE= no transcription


Specificity of hre dna binding domain interaction

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 bindingGRE bindingLigand

DBD HBD

WT-ER

yesno estradiol

HBD

WT-GR

noyes cortisol

HBD

noyesestradiol

ER/GR


How does binding of activated steroid receptor to hres enhance transcription

intron

5’-flanking region

exon

ERE TATA

BOX

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


Do steroids always act through classical steroid receptor mechanisms

Ca++

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


Interactions of steroid receptors with regulatory elements of other signaling pathways

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

(-)

dimerization

Glucocorticoid

Receptor

AP1

collagenase

Molecular Mechanism through which Glucocorticoids inhibit Inflammatory Responses


Inhibition of ap 1 by gr

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

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 progesterone1

Female

Lordotic

Response

C P D1 D1

+

RU486

Activation of Progesterone Receptor by Dopamine in Absence of Progesterone

-O’Malley


Use of antisense oligonucleotides to block gene expression

complementary

DNA sequence

AAAAA..,

PR mRNA

Translation

Start site

mRNA degradation

block translation

Use of Antisense Oligonucleotides to Block Gene Expression

Block Gene Expression


Blockade of dopamine activation of pr by antisense oligonucleotides

Female

Lordotic

Response

C P D1 D1

+

antisense

oligo to PR

Blockade of Dopamine Activation of PR by Antisense Oligonucleotides


Estrogen phosphorylates creb via the mapk pathway wade dorsa 2003

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 20031

E2:ER

E2

MAPK pathway

gene transcription

(CRE)

phosphorylation of CREB

Estrogen Phosphorylates CREB via the MAPK Pathway – Wade & Dorsa, 2003


Regulation of steroid hormone receptor expression

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 expression1

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

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

Next Time…

First Midterm Exam


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