Outline

Nuclear Receptors:
Estrogen Receptor & SERMs

Type I Nuclear Receptors:

steroid receptors
receptor is homodimer
GR Glucocorticoid R
MR Mineralocorticoid R
ER Estrogen R
PR Progesterone R
AR Androgen R

binds to DNA Inverted Repeat (palindromic)

Located in cytoplasm in absence of ligand;

ligand binding causes dissociation of heat
shock proteins, receptor dimerization, and
translocation to nucleus; then binds to DNA
and recruits coactivators

Hormone Response Elements (DNA binding sites for nuclear receptors):

Glucocorticoid
Response Element:

Nuclear Receptor structure

Estrogen receptor and Selective Estrogen
Receptor Modulators (SERMs)

Type II Nuclear Receptors:

non-steroid receptors
receptor is Heterodimer
Usually RXR (retinoid X (9 cis
retinoic acid) receptor) with:
TR (Thyroid hormone R)
VDR (Vitamin D R)
RAR (Retinoic acid R)
PPAR (Peroxisome proliferatoractivated R)
binds to DNA Direct Repeat
Repeats are separated by 1-5
nucleotides
Located in nucleus in absence of ligand,
often complexed with corepressors

Estrogen
Response Element:

Ligand binding to NR causes

dissociation of corepressor and
recruitment of coactivator proteins.

Chromosome Immunoprecipitation (ChIP):

Another method for Protein-DNA analysis
Used for:
mapping the DNA target of
DNA-binding proteins
mapping the DNA target of
histone modifying enzymes

How do structurally similar NR proteins regulate

different genes in different cells?
Ligand:
Different specificity for ligand binding by NR
Metabolic control of ligand availability

Method:
Cross-link the DNA & protein (or
leave in its native bound form)
Fragment the chromatin
Precipitate the DNA-Protein
complexes with antibody to the
protein of interest
Analyze the bound DNA

Nuclear Receptor
Recognize different DNA sequences (hormone
response element)
Different expression patterns of NRs in cells
Activate different coactivators/coregulators

Does ligand specificity explain receptor specificity?

The worst case scenario - compare MR and GR

How do tissues
distinguish
between Cortisol
and Aldosterone?

Glucocorticoid
Mineralocorticoid

Studying Nuclear Receptor Function:

How do tissues distinguish between Cortisol and Aldosterone?

Glucocorticoid Response Element and Mineralocorticoid Response Elements
are identical.
GR and MR both bind glucocorticoids & minerocorticoids with similar affinity
[Cortisol] is typically ~10x higher than [Aldosterone].

inactive
11 hydroxysteroid
dehydrogenase

Chloramphenicol Acetyl Transferase

reporter gene assay measures
acetylation of chloramphenicol

Luciferase reporter gene assay measures

luminescent light emission catalyzed by the
firefly enzyme luciferase

Luciferase

Enzyme located in collecting

duct of the kidneys

Luciferin (substrate)

Homology among Two Classes of Nuclear Receptors

Functional Domains of Nuclear Receptors

Class I

Class II

Functional Domains of Nuclear Receptors

DNA-binding Domain of Nuclear Receptor: Zinc Fingers

Dimerization
Region C

DNA-binding
Region

Zn

Zn
C

Cartoon view of Zinc fingers of estrogen receptor

Ligand Binding Domain of Nuclear Receptor

(Ligand-Binding Domain of estrogen receptor is shown)

Ligand-binding Domain of Estrogen Receptor

(side view)

No Agonist bound
(or Antagonist bound)

Agonist Bound:

Agonist bound

Co-activator binds here

Helix 12
agonist

Antagonist Bound:

Co-activators cant bind,

Instead co-repressors bind
Helix 12

Co-repressors recruited
Histones deacetylated
Transcription repressed

antagonist

Estrogen Effects

Co-activators recruited
Histones acetylated
Transcription enhanced

September 24, 2002

Womens Health Initiative

study on HRT stopped
Estradiol

LH and FSH stimulate estrogen and progesterone production in

ovaries
Reproductive function: ovulation, menstrual cycle, pregnancy
Development of female characteristics in puberty
Bone health, prevention of osteoporosis
amount of good HDL cholesterol; bad LDL cholesterol; risk
of heart disease
Estrogen-dependent cancers (breast, ovarian); cell proliferation

In July, the National Institutes of

Health (NIH) halted a large, in-progress
study examining the effects of a widely
used type of hormone replacement
therapy (HRT) medication called
Prempro, which combines the
hormones estrogen and progestin.
The study, which is one of five major
studies that comprise the large clinical
trial called the Womens Health Initiative
(WHI), was discontinued because the
hormones appear to increase a womans
risk of breast cancer as well as heart
disease, blood clots and stroke.

Thrombosis (blood clots)

Prevent menopausal symptoms (hot flashes, mood swings, fatigue)

Estrogen Receptor
Drugs that bind to Estrogen Receptor

Agonist: Estrogen
Contraceptive
Hormone replacement therapy

Estrogen-Induced Proliferation and

Spontaneous New Mutations
Estrogen stimulation
Normal breast or uterine cell

Selective Estrogen Receptor Modulators (SERM)

partial agonist/antagonist
Clomiphene

Infertility treatment; blocks estrogen binding in anterior pituitary GnRH stimulates ovulation

Tamoxifen

Used to treat estrogen-dependent breast cancer

Raloxifene

Used to treat osteoporosis for post-menopausal women

estrogen-dependent breast cancer and uterine cancer

Mistake in DNA duplication

Antagonist
Fulvestrant

Used to treat metastatic estrogen-dependent breast cancer

Drugs that influence Estrogen Synthesis:

Aromatase Inhibitors
Blocks synthesis of estrogen in post-menopausal women
Used in treatment of breast cancer and ovarian cancer in post-menopausal women

Increased proliferation

Antiestrogens

Aromatase Inhibitors

Coactivator
binds

Estradiol is produced in:

Ovaries
Adrenal cortex (downstream of DHEA), adipose tissue, (and liver,
brain, placenta)
Aromatase enzyme converts androgens to estrogens in peripheral
tissues (e.g. breasts)
Aromatase inhibitors block estrogen production in post-menopausal
women, where major source of estrogen is adrenal cortex
Aromatase inhibitors not generally used in premenopausal women,
because its effects are reduced by hypothalamus/pituitary feedback:
estrogen GnRH LH & FSH estrogen

Estrogen

Antiestrogen

Estrogen
receptor

Estrogen
receptor

Binding
to DNA

Genes are
activated

Binding
to DNA

Coactivator
cannot bind to
antiestrogenbound receptor

No gene
activation

Selective Receptor Modulator (SRMs)

SRMs are partial agonists/antagonists whose action
depends on the ligand and the target tissue.

Tamoxifen and Breast Cancer Prevention

Invasive breast cancer rates in women at high risk
40
Placebo

SERMs: ER (estrogen receptor)

Used for protection of bones & as anti-cancer agents

SPRMs: PR (progesterone receptor)

Under investigation for endometriosis & uterine fibroid tumors

SARMs: AR (androgen receptor)

Develop drugs to protect bones and promote muscle without
side effects?

SGCRM: GR (glucocorticoid receptor)

Develop an anti-inflammatory agent with fewer side effects?

Other SRMs

The Need for Better SERMs

30
Cumulative
number
of cases 20
(per 1000
women)
Tamoxifen

10

0
0

3
Years

Action of SERMS in Target Tissues

Tamoxifen

Fulvestrant

Good effects
Reduces breast cancer risk
Lowers LDL cholesterol
Strengthens bones

Bad effects
Increases uterine cancer risk
Increases blood clot risk

Search for the Perfect SERM

How can SERMs have different

effects on different tissues?
Different estrogen receptors. There are two estrogen receptors: ER
and ER. They are both widely distributed but with some selectivity
for particular tissues. Different SERMs may interact selectively with
different ERs.

The ideal SERM would:

Strengthen bones
Lower LDL cholesterol and
raise HDL cholesterol

ERs may adopt multiple conformations, depending on the ligand,

and recruit different co-activators or co-repressors. A combination of
multiple ER conformations with different tissue distribution of coactivators and co-repressors may be the underlying reason.

NOT increase blot clot risk

Relieve hot flashes
Reduce breast cancer risk

Different distribution and abundance of co-activators and corepressors in different tissues

Reduce uterine cancer risk

For the future: Can new and better SERMs be designed with the
desired agonist/antagonist properties in different target tissues?

Pharmacology of NR Ligands
SERM-bound ER-ligand
binding domains (LBDs) take
intermediate structure

Difference in cofactor binding

surface on these LBDs gives
them distinct preference of
coregulator partners

Model: Relative concentration

of coactivator /corepressor may
determine SERM-bound ERs
action

This model proposes that

SERMs enable ER to bind
multiple types of coregulators.

Summary

Nuclear receptors contain modular transcription-activation domain, DNAbinding domain, and hormone-binding domain

Nuclear receptors bind DNA as a dimer, with -helix from first zinc finger of
each receptor binding to major groove of DNA to recognize half of the
response element

Binding of agonist recruits co-activators (e.g. HATs), while binding of

antagonist recruits co-repressors (e.g. histone deacetylases)

Depending on the ligand and the tissue, either agonist or antagonist effects
may be produced

SRMs provide opportunities to exploit more beneficial effects of nuclear

receptors while avoiding undesired ones.

Selective Estrogen Receptor Modulators (e.g.Tamoxifen, Raloxifene) aim to be

estrogen antagonists in breast & endometrium (to cancer),
antagonists in blood (to stroke) and
estrogen agonists in other tissues (to maintain bone mass, hot flashes, good
HDL cholesterol)