Transcript 4.5 Regulation and Variability of Signaling by Nuclear Receptors

Gerhard Krauss
Biochemistry of Signal Transduction and Regulation(3rd
Edition) ISBN: 3-527-30591-2
LOGO
Signaling by Nuclear
Receptors
授課老師: 褚俊傑副教授 (生物科技系暨研究所)
聯絡電話: 0986-581835
電子信箱: [email protected]
Nuclear Receptor
Signaling
Pathways
Outline
 4.1 Ligands of Nuclear Receptors
 4.2 Principles of Signaling by Nuclear Receptors
 4.3 Classification and Structure of Nuclear
Receptors
 4.4 Mechanisms of Transcriptional Regulation by
Nuclear Receptors
 4.5 Regulation and Variability of Signaling by
Nuclear Receptors
 4.6 The Signaling Pathway of the Steroid Hormone
Receptors
 4.7 Signaling by Retinoids, Vitamin D3, and the T3Hormone
4.1 Ligands of Nuclear Receptors
 The naturally occurring ligands of nuclear receptors
are lipophilic hormones, among which the steroid
hormones, the thyroid hormone T3, and derivatives
of vitamin A and D have long been known as central
regulators.
 These hormones play a significant role in metabolic
regulation, organ function, and development and
differentiation processes. Following formation and
secretion in specific tissues, the endocrine organs,
the hormones are distributed in the organism via the
circulation and enter cells passively by diffusion.
4.1 Ligands of Nuclear Receptors
 In recent years it has been recognized that
intracellularly formed lipophilic metabolites can also
serve as ligands for nuclear receptors and can
regulate gene expression through their binding to
nuclear receptors.
 These
compounds
include
prostaglandins,
leukotrienes, fatty acids, cholesterol derivatives, bile
acids, and even benzoates. The most important
natural ligands of the nuclear receptors are shown in
Fig. 4.1; the cognate receptors and their DNA
elements are summarized in Table 4.1.
Fig. 4.1 Natural ligands of
nuclear receptors.
Tab. 4.1 Ligands and structure of HREs of selected nuclear receptors from
mammals.
Abbreviations: IR: “inverted repeat”; DR: “direct repeat”; ER: “everted repeat”; NR: “no repeat”.
Numbers of HREs give the number of pairs separating the half-sites. R: purine. Y: pyrimidine. NGFI:
Nerve growth factor induced receptor; COUP-TF: CHicken ovalbumin upstream promoter
transcription factor; ROR: Retinoic acid related orphan receptor. a, b und c are receptor subtypes
coded by distinct genes.
4.2 Principles of Signaling by Nuclear
Receptors
 Signal transduction by nuclear receptors is shown
schematically in Fig. 4.2. The nuclear receptors are
localized in the cytosol and/or nucleus. Many of the
natural ligands of nuclear receptors are lipophilic
hormones that enter the cell in a passive manner or by
active transport mechanisms.
 A transmittance of the signal at the cell membrane is not
necessary, as is the case in the signaling via
transmembrane receptors. Once inside the cell, the
hormone ligand binds the cognate receptor which is
localized in the cytosol and/or in the nucleus. The
hormone binding activates the transcription regulation
function of the receptor.
 In the case of the cytosolic receptors, the hormone
binding induces translocation into the nucleus where the
hormone-receptor complex binds a cognate DNA element
termed hormone-responsive element, HRE, and alters the
Fig. 4.2 The principle of signal
transduction by nuclear receptors.
Nuclear receptors are ligand
controlled transcritpion factors
that
bind
cognate
DNA
sequences,
or
hormone
responsive elements (HRE). The
hormone acts as a regulating
ligand. Most nuclear receptors
bind their cognate HRE, which
tend
to
be
symmetrically
organized,
as
homoor
heterodimers. The DNA bound,
activated receptor stimulates
transcription initiation via direct
or
indirect
protein-protein
interactions with chromatin and
the
transcription
initiation
complex.
The
arrows
demonstrate
the
different
possible configurations of the
HRE (see also Section 4.3.1). H:
4.3 Classification and Structure of Nuclear
Receptors
 4.3.1 DNA-Binding Elements of Nuclear Receptors,
HREs
 4.3.2
The
Receptors
DNA-Binding
Domain
of
Nuclear
 4.3.3 HRE Recognition and Structure of the HREReceptor Complex
 4.3.4 Ligand-binding Domains
 4.3.5
Transactivating Elements of the Nuclear
Receptors
4.3 Classification and Structure of Nuclear
Receptors
 The nuclear receptors display a high degree of homology
at the level of amino acid sequence, which indicates that
they operate with similar functional principles.
 For some receptors the cognate hormone and their
function in the cell remain unknown. Such “orphan
receptors” are usually identified by sequence homology
and with the help of screening techniques employing
DNA probes based on known receptors. Possibly some of
the orphan receptors do not have natural ligands and
function in a ligand-independent manner.
 At the level of the primary structure the steroid hormone
receptors can be divided into five different domains (Fig.
4.3), each with specific functions.
Fig. 4.3 Domain structure of the nuclear receptors. Functional domains of
nucear receptors are portrayed in a one dimensional, linear fashion.
4.3.1 DNA-Binding Elements of Nuclear Receptors,
HREs
 The steroid hormone receptors are sequence-specific
DNA-binding proteins whose cognate DNA elements are
termed “hormone-responsive elements” (HREs).
 The identity of an HRE is determined by the sequence,
polarity, and distance of the hexamers. Mutation and
duplication of an ancestral recognition sequence have
allowed the creation of many and various DNA elements
during the course of evolution, whose sequence, polarity
and distance is characteristic for a given hormone
receptor or receptor pair.
4.3.1 DNA-Binding Elements of Nuclear Receptors,
HREs
 The half-site of an HRE can be arranged as a palindrome,
an inverted palindrome or a direct repeat. For a given
receptor, optimal spacings of the half-sites exist, and the
number of base pairs between the half-sites is another
charactistic feature of a HRE. Figure 4.4 illustrates, on the
example of the HREs for the RXR heterodimer, the
various configurations of an HRE.
Fig. 4.4 HRE structure of the
RXR heterodimer. Shown is the
consensus sequence of the
HREs of the RXR heterodimers
(see Fig. 4.7) and the different
possible arrangements of the
hexameric half-site sequences.
The hexamers can be arranged
palindromically
as
inverted
repeats (a), as everted repeats
(b), or as direct repeats (c). n
indicates the number of base
pairs that lie between the two
hexamers. RXR: receptor for 9cis-retinoic acid; RAR: receptor
for all-trans retinoic acid; T3R:
receptor for the T3 hormone;
PPAR peroxisome proliferator
activated
receptor;
VDR:
receptor for vitamin D3.
4.3.1 DNA-Binding Elements of Nuclear Receptors,
HREs
 Based on the subunit structure of DNA-bound
receptors and on the structure of the HREs, four
classes of nuclear receptors can be distinguished
(Fig. 4.5).
 Dimers of the Steroid Hormone Receptors
The HREs of the steroid hormone receptors possess
a palindromic structure, comparable to the DNAbinding elements of procaryotic repressors (see Fig.
4.5a).
 Heterodimers Containing RXR
The DNA-binding elements of the nuclear receptors
for all-trans retinoic acid, for 9-cis retinoic acid, for
the T3 hormone and for the vitamin D3 hormone
usually exhibit a direct repeat of the recognition
sequence, resulting in the formation of heterodimers
4.3.1 DNA-Binding Elements of Nuclear Receptors,
HREs
 Dimeric Orphan Receptors
The “orphan receptors” derive their name from the
fact that the cognate hormones for these receptors
were originally unknown or little understood. Orphan
receptors can bind as homodimers to recognition
sequences arranged as direct repeats (Fig. 4.5c).
 Monomeric “Orphan Receptors”
Orphan receptors are also known (e.g., Retinoic Z
receptor, RZR; NGF-induced clone B, NGFI-B) which
bind as monomers to asymmetric recognition
sequences (Fig. 4.5d).
Fig. 4.5 Oligomeric structure of nuclear
receptors and structure of the HREs.
The
nuclear
receptors
can
be
subdivided into four groups based on
strucutres of the receptors and HREs.
Shown above are some representative
examples. a) binding of a homodimeric
receptor to a two-fold symmetric
palindromic
DNA
element,
GR:
gluccocorticoid receptor. b) binding of a
heterodimeric receptor to a DNA
element with direct repeats of the
recognition sequence, whereby the 5’
side of the HRE is occupied by the
receptor for 9-cis retinoic acid (RXR).
RAR: receptor for all-trans retinoic acid,
T3R: receptor for the T3 hormone; PPAR:
peroxisome
proliferating
activated
receptor; VDR: receptor for vitamin D3.
c) binding of RXR as a homodimer to an
HRE with direct repeat of the
recognition sequence. d) binding of a
monomeric receptor to an asymmetric
recognition sequence, NGFI-B: nerve
growth factor induced receptor, is
involved in the regulation of enzymes of
4.3.2 The DNA-Binding Domain of Nuclear
Receptors
 The binding of receptors to their cognate HRE
occurs via a DNA-binding domain, which are largely
independent folding domains. Within the family of
nuclear receptors, the DNA-binding domain is the
most conserved structural element and is located in
region C of the primary structure (see Fig. 4.6).
 The DNA-binding domain possesses structural
elements that mediate the specific recognition of the
HRE, as well as for the dimerization of the receptor
on the HRE. The core of the DNA-binding domain
includes a span of 70–80 amino acids, in which all
information for the specific recognition of the
cognate half-site is contained.
Fig. 4.6 Functional domains,
DNA binding and HRE structure
of nuclear receptors on the
example
of the glucocorticoid receptor,
GR. a) domain structure of GR.
AF1, AF2: domains that mediate
the
stimulation
of
the
transcription.
b)
schematic
representation of the two Zn2+Cys4 binding motifs of the DNA
binding domains. c) Complex
formation between the dimeric
DNA binding domains of GR and
the HRE. The black spheres
represent
Zn2+
ions.
d)
Consensus
sequence
and
configuration
of
the
HRE
elements of GR.
4.3.3 HRE Recognition and Structure of the HRE-Receptor
Complex
 For direct repeat HREs, the spacing of the two half-sites
is often the decisive, if not the only, element based on
which the receptor (homodimer or heterodimer)
recognizes its own HRE and discriminates against
related HREs.
 The solution of the structures of heterodimer-DNA
complexes has shown how these receptors can
distinguish between highly related HREs (Fig.4.7). As an
example, the structure of a DNA-bound receptorheterodimer composed of the DNA-binding domain of
RXR and the T3-receptor is given in Fig. 4.7.
Fig. 4.7 Structure of the RXR-T3R heterodimer in complex with DNA. Illustrated is a
complex between the DNA binding domain of the RXR-T3R heterodimer and an HRE
with direct repeats of the sequence AGGTCA separated by 4 base pairs. The two
receptor subunits contact the hexameric sequences with a recognition helix in a
manner very similar to that of the gluccocorticoid receptor (see Fig. 4.6). The Zn atoms
are drawn as green spheres. The figure illustrates the polarity of the binding of the two
subunits. The interaction between the two subunits is mediated mainly via an
extension of the C-terminal DNA binding domain (bottom) of the T3R. A greater or
smaller distance between the two hexamers of the HRE would act contrary to the
interaction between the two subunits as shown.
4.3.4 Ligand-binding Domains
 The region E with the ligand-binding domain harbors
three important functions:
– homo- and heterodimerization
– binding of ligand, both agonists and antagonists
– transactivation and transrepression: binding of
coactivators and corepressors.
 Dimerization
A contribution to the dimerization of the receptors – in
addition to that from the DNA binding domain – is
provided by a dimerization element in the ligand-binding
domain. The structure of the ligand-binding domain of
RXR without bound hormone shows a homodimer with a
symmetric dimerization surface, formed essentially from
two antiparallel a-helices.
4.3.4 Ligand-binding Domains
 Ligand Binding
The crystal structures of the ligand-binding domains of several nuclear
receptors have been resolved demonstrating a similar overall
structure. Fig. 4.8a shows the receptor for alltrans retinoic acid, RAR,
with is bound ligand. The ligand-binding domain is formed from 12 ahelices numbered from H1 to H12. In the bottom half of the structure, a
ligand-binding pocket is found, which accommodates the ligand.
The pocket is mainly hydrophobic and of variable size for different
receptors. As illustrated in Fig. 4.8b for the binding of RAR bound to
all-trans-retinoic acid, contacts between the ligand and the pocket can
be very extensive and include many hydrophobic contacts as well as
hydrogen bonds to the polar parts of the ligand.
Fig. 4.8 a) Ribbon diagram of
RAR bound to all trans retinoic
acid. b) Schematic diagram of
retinoic acid binding site. Hbonds and ionic interactions fix
the carboxylate of the ligand
whose nonpolar parts are deeply
buried in a hydrophobic pocket.
4.3.4 Ligand-binding Domains
 Ligand Binding
The H12 helix is amphipathic, possessing a hydrophobic and a
hydrophilic face. In the unliganded RXR, helix 12 projects away from
the body of the ligand-binding domain. In the liganded structure, the
helix reorients (Fig. 4.9).
As illustrated in Fig. 4.10, the structure of a ligand-binding domain with
an antagonist bound can provide a rational basis to explain the
antagonistic function of a ligand.
Fig. 4.9 Structural changes in the ligand
binding domain of RXR on binding of 9cis retinoic acid. The models of domain
E of apo-RXRα and the binary complex
of RXRα and 9-cis retinoic acid were
superimposed. Domain E of apo-RXRα is
depicted in green and yellow, and
domain E of the binary complex in blue
and red. The grey arrows indicate the
structural rearrangements of helices 11,
12, and the N-terminus of helix 3.
Fig.
4.10
Agonist
vs
antagonist
binding
to
estrogen receptor, ER a)
Ribbon diagram of ER
bound to the agonist
estradiol (black). b) Ribbon
diagram of ER bound to the
antagonist
raloxifene
(black).
c)
schematic
drawing
of
estradiolbinding site. d) schematic
diagram
of
raloxifenebinding site.
4.4 Mechanisms of Transcriptional Regulation by Nuclear
Receptors
 Most of the functions of nuclear receptors can be
described in terms of activation and repression of
transcription. Although attention has been focussed
primarily on the transcription activation mediated by
“positive” HREs, it is increasingly recognized that nuclear
receptors can also repress transcription in a liganddependent manner.
4.4 Mechanisms of Transcriptional Regulation by Nuclear
Receptors
 So-called “negative HREs” have been identified that bind
the receptor and mediate negative regulation by the
ligand. These elements have been identified for
glucocorticoid receptors and for the T3 receptor.
 In addition to ligand-dependent activation and
repression, a subset of nuclear receptors repress basal
transcription in the absence of ligand when bound to a
positive HRE, thereby silencing the target gene (Fig.
4.11).
Fig. 4.11 Model of repression and activation of T3R. In the absence of the T3 hormone, a
heterodimeric RXR-T3R receptor is bound at the T3-responsive element,TRE, establishing a basal
repressed state. The repressed state is maintained by recruitment of corepressor complexes
containing histone deacetylase activity. X refers to potential unidentified cofactors (possibly
chromatin remodeling complexes or SRBs) which help to keep the promotor-bound basal
transcription apparatus in the inactive state. In the presence of T3 hormone, the corepressors are
removed and coactivators (e.g. the SRC/p160 complex) bind to the receptor heterodimer. The
histone acetylase activity of the associated proteins helps to induce a transcription competent state
of the nucleosomes. Furthermore, another coactivator complex, DRIP/TRAP, is recruited by to the
promotor which is thought to stabilize binding of the RNA polymerase holoenzyme.
4.4 Mechanisms of Transcriptional Regulation by Nuclear
Receptors
 Coactivators of Nuclear Receptors
A series of proteins or protein complexes with
coactivator function for nuclear receptors has been
identified that specifically interact with the activated,
liganded receptor. The most abundant ones are now
included in the p160 family of coactivators with the
steroid receptor coactivator 1, SRC-1, as a wellcharacterized member.
Another group of coactivators is present as multiprotein
complexes like the TRAP complex (TRAP, thyroid
hormone receptor activating protein). A common
receptor interaction motif LXXLL is found on these
coactivators which mediates at least part of the
interaction with the AF2 domain of the receptor.
4.4 Mechanisms of Transcriptional Regulation by Nuclear
Receptors
 Corepressors of Nuclear Receptors
Unliganded T3 receptor (T3R) and retinoic acid receptor
(RAR) can repress transcription in the absence of ligand.
This transcriptional repression is linked to the binding of
proteins with corepressor activity. Examples of such
corepressors are the nuclear corepressor NcoR and the
“silencing mediator for retinoic and thyroid hormone
receptors”, SMRT.
Both proteins interact with the ligand-binding domain
and appear to be released from the receptor upon ligand
binding. Possibly alternative conformations of the AF-2
region serve to trigger this release.
4.5 Regulation and Variability of Signaling by Nuclear
Receptors
 Signaling by nuclear receptors is regulated at
various levels (Fig. 4.12). The following are important
regulatory attack points:
•
•
•
•
Regulation at the level of ligand concentration
Crosstalk: regulation by phosphorylation
Interaction with other transcriptional activators
Regulation by ubiquitination
Fig. 4.12 Functions of nuclear receptor domains. The domains A/B, C, E and F
of the nuclear receptors are involved in multiple protein-protein interactions
and are subject to regulatory modifications as indicated. Most important are
the corepressor and coactivator complexes that direct histone deacetlyase
(HDAC) and histone acteylase (HAT) activities, respectively, to the nuclear
receptor regulated promotor region. TRAP: thyroid hormone receptor
activating protein.
4.5 Regulation and Variability of Signaling by Nuclear
Receptors
* Regulation at the level of ligand concentration
A main determinant of nuclear receptor signaling is the
concentration of the ligand available for binding. The ligand
concentration can be regulated in many ways (for details
see textbooks on hormone action):
– synthesis and degradation
– modification
– secretion, transport and storage
– feedback regulation via the circulating hormone
concentration
4.5 Regulation and Variability of Signaling by Nuclear
Receptors
* Crosstalk: regulation by phosphorylation
The phosphorylation of nuclear receptors on Ser/Thr
residues is a key mechanism for the coupling of nuclear
receptor signaling to other signaling pathways of the cell.
Many nuclear receptors are isolated as phosphoproteins,
and their phosphorylation provides a means for ligandindependent activation and regulation.
4.5 Regulation and Variability of Signaling by Nuclear
Receptors
* Interaction with other transcriptional activators
Nuclear receptors can also modulate gene expression by
interference with the activity of other transcriptional
activators. The ER, T3R, RAR and GR proteins have, e.g.,
been shown to act as transrepressors of the transcription
factor AP-1, which is a heterodimer composed of c-Jun and
c-Fos
proteins.
Reciprocally,
AP-1
can
inhibit
transactivation by these receptors.
* Regulation by ubiquitination
As for other central regulators, the level of nuclear
receptors is modulated by ubiquitin-mediated degradation.
A ligand-dependent ubiqutination and subsequent
proteasomal degradation has been described for ERa, PR,
VDR, T3R and RARa, allowing for a down-regulation of
signaling by these receptors.
4.6 The Signaling Pathway of the Steroid Hormone
Receptors
 Based on the receptor activation mechanism, the nuclear
receptors may be divided into two basic groups. In the
first group (those including most of the steroid hormone
receptors), the receptors can be localized in the nucleus
or in the cytoplasm.
 Signal transduction by steroid hormones is distinguished
by the fact that the receptors can be found either in the
cytoplasm or in the nucleus. The steroid hormone
receptor receives the hormonal signal in the cytosol,
becomes activated by hormone binding, at which point it
enters the nucleus to regulate the transcription initiation
of cognate genes.
 Figure 4.13 shows the most important steps in the signal
transduction by steroid hormones.
Fig. 4.13 Principle of signal
transduction by steroid hormone
receptors. The steroid hormone
receptors in the cytosol are found
in the form of an inactive complex
with the heat shock proteins Hsp90
and Hsp56 and with protein p23.
The binding of the hormone
activates the receptor so that it can
be transported into the nucleus
where it can bind to its cognate
HRE. It remains unclear in which
form the receptor is transported
into the nucleus, and to which
extent the associated proteins are
involved in the transport. One
mechanism
of
activation
of
transcription initiation involves
mediation by the proteins RIP-140
and Sug1.
4.7 Signaling by Retinoids, Vitamin D3, and the T3Hormone
 4.7.1
Structure of the HREs of RXR
Heterodimers
 4.7.2 Complexity of the Interaction between
HRE, Receptor and Hormone
4.7 Signaling by Retinoids, Vitamin D3, and the T3Hormone
 In contrast to signal transduction by the steroid hormone
receptors, there are multiple pathways by which the
ligands of this group are made available for receptor
activation (Fig. 4.14):
* The hormone ligands can be secreted in the classical
endocrinological pathway and transported to the target
cell where they bind the receptor.
* The active hormones can be formed intracellularly from
inactive precursors. The inactive precursor is transported
through the bloodstream to the target cell where it is
enzymatically converted to the active hormone. An
example of this pathway is that of 9-cis retinoic acid,
which is synthesized from the alcohol of vitamin A
(vitamin A1; retinol).
Fig. 4.14 Principle of signal transduction by RXR heterodimers. The activated hormone
can be made available to the RXR heterodimer in three different ways. a) The hormone
(e.g. T3 hormone) is synthesized in endocrinal tissue and reaches the DNA bound RXR
heterodimer in the nucleus via passive transport. b) The active hormone is formed in
the cytosol from an inactive apo-hormone (as for, e.g. 9-cis-retinoic acid). c) The
hormone is synthesized intracellularly. In all three cases, the binding of the hormoneRXR-heterodimeric complex is the signal that induces transcription activation of the
downstream genes.
4.7.1 Structure of the HREs of RXR Heterodimers
 The following points were identified as important for
the recognition and discrimination of a particular
HRE:
• In the case of identical hexamer sequences of an HRE,
the spacing between the hexamers is a specificitydetermining element (n-rule). The spacing can be
between 1 and 6 bp. Grounds for the discrimination
based on spacing is the structure of the receptor dimer.
A given receptor demands a particular spacing of the
hexamers in the HRE because of steric requirements.
• The receptor for 9-cis retinoic acid (RXR) usually
occupies the 5’ position in the heterodimer. The RXR
serves as a quasi vehicle to bring other receptor
monomers to the 3’ half-site of the HRE.
4.7.2 Complexity of the Interaction between HRE,
Receptor and Hormone
 There is an altogether complex interaction between
HRE, receptor and hormone in the group of
receptors for retinoids, vitamin D3 and the T3hormone. The complexity is determined by the
following factors:
* Formation of the Homo- or Heterodimer
* Multiplicity of the HREs
* Multiplicity of the Receptors
* Binding and Activation via Hormones
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