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HORMONE ACTION

VIA
NUCLEAR RECEPTORS

Dr. Michal Lahav


HORMONES WHICH ACT VIA NUCLEAR
RECEPTORS (NRs)

While most regulators act via membranal


receptors, some (mostly hormones) have
intracellular receptors.
The ligands of such receptors are small
hydrophobic molecules, and binding of
the appropriate ligand converts them into
active transcription factors, namely,
proteins which stimulate, or reduce, the
rate of gene transcription.
HORMONES WITH NUCLEAR RECEPTORS (continued)

Thus these receptors, collectively


named nuclear receptors (NRs),
function as ligand-activated
transcription factors.
The five types of steroid hormones
have nuclear receptors, and so do the
thyroid hormones.
In addition, derivatives of vitamin D and
vitamin A (retinoids), which are
synthesized from precursors
consumed as food, also have NRs.
STEROID VERSUS NON-STEROID LIGANDS

The ligands shown in the next slide are


divided into subgroups: steroids (left
column) and non-steroids (middle
column).
The right column shows the names of
the receptors of the compounds
presented.
Some receptors are named after a group
of compounds (natural or synthetic), all
of which can activate that receptor (e.g.,
estrogen receptor), rather than after a
specific compound.
HORMONES WITH NUCLEAR RECEPTORS
11-CIS- RETINAL IN THE RETINA: ACTION NOT
MEDIATED BY A RETINOID RECEPTOR
MECHANISM OF RECEPTOR ACTIVATION

The inactive receptors of four of the


steroids: cortisol, aldosterone,
progesterone and testosterone (GR, MR,
PR and AR) reside in the cytoplasm. A
single receptor molecule forms a complex
with several proteins, most of them heat-
shock proteins (HSPs). The composition
of the complexes of the various receptors
vary somewhat, but all of them include
HSP90.
HSPs are known to be induced by various
cellular stresses, and to act as chaperons,
namely, proteins that act to preserve the
conformation of other proteins.
RECEPTOR ACTIVATION (Continued)

When one of the four steroid hormones


binds to its receptor, the complex
disintegrates; two activated receptor
molecules form a homodimer, which
enters the nucleus and functions as a
transcription factor.
The inactive estradiol receptor resides
in the nucleus as a dimer, in a complex
with one HSP90 molecule, which
dissociates upon binding of the
hormone.
ACTIVATION OF STEROID HORMONE
RECEPTORS
RECEPTOR ACTIVATION (Continued)

The inactive receptors of thyroid


hormones, vitamin D and the retinoids
reside in the nucleus. Upon hormone
binding they form dimers. TR regulates a
few genes as a homodimer, but in most
cases it acts as a heterodimer with RXR.
VDR and RAR act primarily as
heterodimers with RXR.
In some cases the inactive receptor binds
to promoters and act in an opposite
manner to that of the activated receptor
(inhibition instead of activation).
STRUCTURE OF NUCLEAR RECEPTORS (NRs)
(see explanations below)
STRUCTURE OF NUCLEAR RECEPTORS

Nuclear receptors are homologous to a


variable extent. It is customary to divide
the receptor molecule into five domains:
A/B, C, D, E, and F.
Domain C, the DNA-binding domain,
shows the highest degree of homology
among the receptors.
Domain E, the ligand-binding domain,
shows lower degree of homology.
Greater homology is observed when the
ligands are similar.
STRUCTURE OF NUCLEAR RECEPTORS (Continued)

The transactivation domains, namely,


those involved in affecting the
transcription machinery, are A/B and E (E
is ligand-dependent). They are named
AF-1 and AF-2, respectively.
(AF=activating function).
The above figure shows also the
sequences responsible for other
functions of the receptor: dimerization,
HSP binding, and nuclear localization.
THE TWO ZINC FINGERS IN THE DNA-
BINDING DOMAIN (DBD)
STRUCTURE OF A
HOMODIMER OF THE
DNA-BINDING DOMAIN
OF A NUCLEAR
RECEPTOR:
THE PROXIMAL (N-
TERMINAL) ZINC
FINGERS OF THE
MONOMERS FIT
CONSECUTIVE TURNS IN
THE MAJOR GROOVE OF
THE DNA DOUBLE HELIX.
THE DISTAL ZINC
FINGERS (INCLUDED IN
THE DIMERIZATION
ZONE) LIE OVER THE
MINOR GROOVE.
REGULATION OF TRANSCRIPTION
REGULATION OF TRANSCRIPTION
(PRINCIPLES)

The above figure illustrates the


regulation of transcription in animal
cells. Sections of the plasma
membrane, and the outline of the
nuclear membrane, are shown
schematically.
RNA polymerase II (Pol II), which
executes transcription, is shown as
part of a complex with other proteins
(general transcription factors).
REGULATION OF TRANSCRIPTION (Continued)

This complex, the basal transcription


complex, is bound to a sequence of bases in
the DNA, upstream of the transcription
initiation site. In most genes this DNA site
includes the sequence TATA, and is named
the TATA box.
The basal transcription complex interacts
with various proteins which bind to the
DNA, usually upstream of the polymerase
complex. These proteins (transcription
factors - TFs) regulate (increase or
decrease) the rate of transcription.
REGULATION OF TRANSCRIPTION (Continued)

The promoter of a gene is the DNA


section upstream of the transcription
initiation site, and includes both the
RNA polymerase complex binding site
and the DNA section which binds the
transcription factors (a couple of
thousands basepairs may be relevant).
Transcription factors bind selectively to
certain DNA sequences (response
elements REs).
REGULATION OF TRANSCRIPTION (Continued)

The above figure shows examples of


two types of TFs: ligand-activated (a
steroid hormone receptor), and
phosphorylation-activated (substrates
of protein kinases, which had been
activated as a consequence of
increases in second messengers).
Certain TFs are known to be activated
by dephosphorylation rather than by
phosphorylation.
REGULATION OF TRANSCRIPTION (Continued)

Usually, transcription factors affect the


Pol II complex indirectly, with the
mediation of proteins named
coactivators or corepressors,
depending on the effect on transcription.
The figure shows a mechanism in which
the steroid hormone receptors need two
coactivators in a row.
The second coactivator (CBP) mediates
also the action of other TFs.
REGULATION OF TRANSCRIPTION (Continued)

Coactivators are activated allosterically


by TFs and in turn affect allosterically
proteins down the chain.
Some coactivators (in the figure: CBP)
also stimulate transcription by another
mechanism: they have enzymatic
activity of histone acetyl transferase
(HAT). Acetylation of certain lysine
residues in histones loosens the tight
chromatin structure, thus facilitating
transcription.
REGULATION OF TRANSCRIPTION (Continued)

Similarly, corepressors exert allosteric


activation on histone deacetylases
(HDACs), which have an opposite effect
on transcription.
RESPONSE ELEMENTS FOR THE
STEROID HORMONE RECEPTORS

ACTGGA
PALINDROME
(INVERTED REPEAT)
AGGTCA TGACCT

DIRECT REPEAT
AGGTCA AGGTCA
RESPONSE ELEMENTS OF NRs

As shown in the above figure, dimers of


NRs bind to response elements
comprised of two half-sites; each half-site
binds one monomer.
One type of response element is a
palindrome (inverted repeat), in which the
half-sites, one a mirror image of the other
regarding the sequence of bases, are
found in each DNA strand. This is the
most common response element for
steroid hormone receptor homodimers.
RESPONSE ELEMENTS OF NRs (continued)

The other type of response element is a


direct repeat, in which the same base
sequence appears twice in a row on the
same DNA strand.
The specificity of a response element
for a receptor is determined not only by
the base sequence but also by the
spacing between the two half-sites.
It should be stressed that TFs other than
NRs are often homo- or heterodimers,
and their response elements are also of
the above types.
ADDITIONAL MODES OF ACTION OF
TRANSCRIPTION FACTORS

As described above, many TFs act via direct


binding to the DNA. However, an alternative
mode of action is binding by protein-protein
interaction to another TF, which in turn is
bound to the DNA. In such case, the TF
which does not bind to DNA directly may
either augment or suppress the action of the
DNA-bound TF.
Another mode of action is the binding of a
TF to another TF, thus excluding the
association of the latter with the DNA.
ADDITIONAL MODES OF ACTION OF
TRANSCRIPTION FACTORS (Continued)

Some TFs can act via two of the described


mechanisms, or even by all three of them.
This pertains to some nuclear receptors.
Most of the actions of the glucocorticoid
receptor (GR) to suppress the immune
system involve protein-protein interaction
with other TFs (second and third
mechanism).
The estrogen and androgen receptors were
also shown to bind to DNA indirectly (via
other TFs) in some promoters.
ADDITIONAL MODES OF ACTION OF
TRANSCRIPTION FACTORS (Continued)

When the activated receptors of steroid


hormones act on transcription via
protein-protein interaction, they act as
monomers rather than as dimers.
ORPHAN RECEPTORS

After several nuclear receptors had


been cloned, investigators looked for
additional homologous proteins at the
RNA level. Most of the novel NRs
found had no known ligands, and thus
were named orphan receptors.
Eventually, ligands were identified for
some orphan receptors. These ligands
included regulatory molecules arriving
from the outside, as well as metabolites
synthesized within the cell.
ORPHAN RECEPTORS (Continued)

The ligands identified for orphan receptors


are hydrophobic, as are the ligands of the
classical NRs.
Some of the orphan receptors may be
regulated by phosphorylation rather than by
ligands.
There is great interest in the orphan
receptors, especially since some drugs
used in clinical medicine, as well as some
pollutants, act via orphan receptors.