Endocrinology Topics
Nuclear Receptors
Nuclear receptors are transcription factors activated by binding their cognate ligands. Ligand binding causes a conformational change that places the C-terminal transactivation domain in an active conformation. Once activated they will dimerize with another of the same nuclear receptor or other dimerization partner. The dimer binds a specific region in the promoter of genes controlled by the receptors, known as the hormone response element (HRE). The active conformation allows recruitment of "transcriptional co-activators" such as SRC-1 (steroid receptor coactivator 1) which help promote transactivation of target genes. Since nuclear receptors act to regulate transcription of target genes, their classical mechanism of action is relatively slow taking 20 minutess to hours to see the final effect.
The nuclear receptor superfamily of transcription factors includes many hormone receptors, as well as receptors for other types of ligands, and orphan receptors for which a ligand is not known. They all have similar structure which includes at least the following domains:
A/B = transactivation domain
(AF1)
C = DNA binding domain (zinc-coordinated)
D = less conserved region
E = ligand binding/dimerization/transactivation domain (AF2)
F = less conserved region
The HRE is organized differently for different receptors in a way that corresponds with the receptor dimerization pattern. The HRE for steroid receptors is palindromic, i.e. consists of inverted repeats, and receptor dimers are arranged as mirror images of each other. RXR heterodimers are arranged in sequence and interact with either direct repeats or everted repeats of the HRE. Dimeric orphan receptors are arranged in sequence and interact with direct repeats of the HRE. Some orphan receptors may also interact with the HRE as monomers.
The HRE usually consists of either AGAACA (for glucocorticoid, mineralocorticoid, progesterone, or androgen receptors) or AGGTCA/AGTTCA (for estrogen, thyroid, retinoid or vitamin D receptors) in sequence separated from each other by either 3, 4 or 5 nucleotides. Here are some examples:
Glucocorticoid Receptor:
5' AGAACAnnnTGTTCT 3'
3' TCTTGTnnnACAAGA 5'
Estrogen Receptor:
5' AGGTCAnnnTGACCT 3'
3' TCCAGTnnnACTGGA 3'
Thyroid Receptor:
5' AGGTCAnnnnAGGTCA
3'
3' TCCAGTnnnnTCCAGT
3'
or
5'
AGGTCAnTGACCT 3'
3' TCCAGTnACTGGA 3'
Many nuclear receptors form dimers with the Retinoid X Receptors (USPs in Drosophila). Before the discovery of the RXRs, it appeared that the RAR, VDR and TR needed partner proteins to work optimally. It was later discovered that RXRs perform that function, e.g. RAR/RXR heterodimers. There are at least three RXRs: RXRa, RXRb, RXRg. They were cloned by homology to the RARs and were considered orphan receptor at first. RXRs work alone as as well, forming RXR/RXR homodimers that bind 9-cis-retinoic acid, an endogenous protein.
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Coactivators and Corepressors
Upon
binding their ligands (often hormones), nuclear receptors, such as the steroid
receptors, undergo a conformational change of the C-terminal transactivation
domain known as AF-2. They then bind to their target DNA sites and recruit co-activator
proteins and general transcription factors.
Transcription factors recruit proteins that increase (or decrease) RNA polymerase activity by making the gene “open” (or “closed”) for RNA polymerase action. They accomplish this by bring in protein enzymes that modify the gene chromatin. Key experiments to understand shuch mechanisms were done with nuclear receptors.
One of the mechanisms by which genes remain unaccessible to RNA polymerase is histone deacetylation. When cells are not growing, their chromatin is tightly packed around complexes of histone proteins called nucleosomes. Acetylation of histones helps loose the DNA from its packaging, making it more accessible for transcription. An example of a co-activator with histone modification activity is SRC-1.
SRC-1
is a transcriptional co-activator for the steroid receptor superfamily. It is
a histone acetyltransferase, thus it modifies chromatin histone proteins to
activate the chromatin and allow gene activation.
Other co-activators include SRC-2/TIF2/GRIP-1 and pCIP/RAC3/ACTR/AIB 1/SRC-3. AIB1 is overexpressed in many breast cancers and may therefore be important in tumorigenesis.
Co-activators also may interact with/recruit other (general) transcriptional co-activators such as P300 and CBP (also histone acetyltransferases that are sometimes known as cointegrators).
A distinct family of proteins may be involved in silencing nuclear receptors in the absence of ligands. These proteins are called "co-repressors" and include N-Cor (nuclear receptor co-repressor) and SMRT (silencing mediator of retinoid and thyroid receptors). N-Cor and SMRT usually dissociate from the receptors when ligand binds. N-Cor and SMRT repress transcription by recruiting histone deacetylases such as mSin3A and mRPD3/HDAC 1.
Some orphans receptors also interact with co-repressors. Interactions with unknown ligands or other proteins may lead to dissociation in these cases.
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Non-Classical Nuclear Receptor Mechanism
Constitutive Androstane Receptor Beta (CARb) binds to DNA as an heterodimer with RXR and activates gene transcription consitutively (i.e. is always on). Stereospecific forms of the androstane metabolites androstanol and androstenol are CARb ligand ( ex. the mammalian pherormone 5a-androst-16-en-3a-ol), but in contrast to most other ligands, they inhibit the activity of CARb. Therefore they are naturally occurring inverse agonists. CARb may be in active conformation in the absence of ligand. Binding of specific inverse agonist ligands may shift the receptor to an inactive conformation, promoting the release of co-activators.
“Rapid” or “non-genomic” effects seem to be mediated by the known “nuclear” receptors acting outside the nucleus or by similar receptors acting outside the nucleus. There are many examples now known for estrogen, progesterone and thyroid hormones. Some rapid estrogen effects are well known: activation of membrane-associated kinases in bone cells and membrane associated nitrous oxide synrhase (NOS) in vascular epithelium, and antiapoptotic action in brain neurons. Classical effects of estradiol include regulation of target genes in uterus, breast and brain, and maintenance of uterine mass.
ANGELS
(Activators of nongenomic estrogen-like signaling) like estren (4-estren-3alpha,
17beta-diol) act on bone within 1-2 minutes by activating protein kinases. The
kinases then can have both have rapid nongenomic effects by phosphorylating
other cytosolic proteins, and slower genomic effects by promoting phosphorylation
of transcription factors (including ERs) [research by Stavros Manolagas, University
of Arkansas Little Rock]. This is still very controversial, but ANGELs such
as estren may work on bone to prevent osteoporosis without hside effects due
to classical genomic estrogen signaling.
Rat oxytocin receptor (a membrane receptor) function is inhibition of by direct binding of progesterone (E. Grazzini et al. 1998. Nature 392, 509-512). This study demonstrates transcription-independent signaling pathways of the steroid hormone progesterone and challenges the conventional view that lipophilic hormones such as steroids enter cells and exert effects only though nuclear receptor-mediated signaling pathways. It was already known that progesterone opposes actions of oxytocine, for example it stops the onset of uterine contractions. Progesterone binds to the OT-R with 20 nM affinity, much less than its affinity for the PR (<1 nM), but is compatible with the very high concentration of progesterone during pregnancy (500 nM).
To show that progesterone can exert its effects at the surface of the cell, it was tethered to a membrane-impenetrable carrier protein. Progesterone binding reduced the number of OT-Rs that were available to bind OT. The OT-R is a GPCR that promotes production of IP3 and intracellular Ca2+, thus it was showed that Ca2+ mobilization was inhibited by progesterone in less than a minute.
The effects of progesterone on the OT-R are specific. Progesterone does not inhibit AVP and its receptor (VR), which is similar to OT-R. Other hormones similar to progesterone - E2, testosterone, dexamethasone, aldosterone - do not have the same inhibitory effect of OT-R. The progesterone antagonist RU486 inhibits (?).
Progesterone does not inhibit human OT-R, but a progesterone derivative, 5b dihydroxyprogesterone, does inhibit the human OT-R.
Other non-genomic/rapid effects of progesterone may include: induction of oocyte maturation, and anesthetic effects (still used in veterinary medicine).
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Nice Pictures
Insect Hormones
Text
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Continue
to "Bone Homeostasis" or take a
test: [T1] [T2] [T3].
Need more practice? Answer the following review questions:
1- What is the basic mechanism of action of nuclear receptors?
2- What is the immediate effect of ligand binding on nuclear receptors?
3- What is the general role of dimers in nuclear receptor signaling?
4- What are transcriptional coactivators?
5- How fast are the classical effects of nuclear receptors seen?
6- List 3 general types of nuclear receptors.
7- What is an orphan receptor?
8- List the 5 basic domains of a nuclear receptor, in order from N-terminus to C-terminus, and describe them briefly.
9- What is the HRE?
10- What is a palindromic DNA sequence?
11- What is the general rule for steroid HREs and dimers?
12- What is the general rule for RXR receptor heterodimers and their HREs?
13- WHat are RXR?
14- List 5 nuclear receptors that form RXR heterodimers.
15- What is the ligand of RXR?
16- What are coactivators?
17- List one mechanism by which coactivators "open" DNA and name a coactivator that works through it.
18- List 3 coactivators.
19- List 2 cointegrators recruited by coactivators.
20 - What are corepressors?
21- List 2 corepressors.
22- List 2 histone deacetylases.
23- What is the CARb and how does it work?
24- List 2 CARb ligands.
Answers:
1- What is the basic mechanism of action of nuclear receptors?
Nuclear receptors are transcription factors activated by binding their cognate
ligands.
2- What is the immediate effect of ligand binding on nuclear receptors?
Ligand binding causes a conformational change that places the C-terminal transactivation
domain in an active conformation.
3- What is the general role of dimers in nuclear receptor signaling?
Most active nuclear receptors will dimerize with another of the same receptor
type or other dimerization partner, before they can bind DNA and effect transcription.
Some nuclear receptors do not need dimerizetion to bind DNA.
4- What are transcriptional coactivators?
Proteins recruited by active nuclear receptors to the DNA transcription machinery,
which help promonte transactivation of target genes.
5- How fast are the classical effects of nuclear receptors seen?
Since the calssical effects of nuclear receptors ar mediated by transcription
of regulated genes, takes between 20 minutes to a few hours to detect the final
effect.
6- List 3 general types of nuclear receptors.
hormone receptors
receptors for ligands other than hormones
orphan receptors
7- What is an orphan receptor?
A nuvclear receptor for which a ligand has not been identified.
8- List the 5 basic domains of a nuclear receptor, in order from N-terminus
to C-terminus, and describe them briefly.
A/B = transactivation domain (aka activation function 1; AF-1)
C = DNA binding domain
D = a less conserved intermediate (aka hinge) region
E = ligand binding/dimerization/transactivation domain (aka activation function
2; AF-2)
F = a less conserved C-terminal region
9- What is the HRE?
Specific region in the promoter a target gene to which a specific nuclear receptor
binds.
10- What is a palindromic DNA sequence?
Consists of inverted repeats of the same DNA equence.
11- What is the general rule for steroid HREs and dimers?
The HRE for steroid receptors consists of palindromic repeats separated by either
3, 4 or 5 other nucleotides.Homodimers are arranged as mirror images of each
other.
12- What is the general rule for RXR receptor heterodimers and their HREs?
RXR heterodimers are arranged in sequence and interact with either direct repeats
or everted repeats of the HRE.
13- WHat are RXR?
Retinoid X receptors, form homodimers or heterodimers with other nuclear receptors.
14- List 5 nuclear receptors that form RXR heterodimers.
thyroid hormone receptor (TR)
all-trans retinoid acid receptor (RAR)
vitamin D receptor (VDR)
PPAR
EcR
15- What is the ligand of RXR?
9-cis-retinoic acid
16- What are coactivators?
Proteins recruited by transcription factors that increase RNA polymerase activity
by making the gene "open" for RNA polymerase action.
17- List one mechanism by which coactivators "open" DNA and name
a coactivator that works through it.
histone deacetylation, SRC-1 (a histone acetyltransferase)
18- List 3 coactivators.
SRC-1
SRC-2/TIF2/GRIP-1
SRC-3/AIB1/pCIP/RAC3/ACTR
19- List 2 cointegrators recruited by coactivators.
P300
CBP
20 - What are corepressors?
Proteins involved in silencing nuclear receptors in the absence of ligand, usually
by recruiting histone deacetylases. They usullly dissociate from receptors when
ligand binds.
21- List 2 corepressors.
nuclear receptor corepressor (N-Cor)
silencing mediator of retinoid and thyroid receptors (SMRT)
22- List 2 histone deacetylases.
mSin3A
mRPD3/HDAC1
23- What is the CARb and how does it work?
Constitutive Androstane Receptor beta binds DNA as an heterodimer with RXR and
activates gene transcription constitutivelly, until deactivated by ligand binding.
24- List 2 CARb ligands.
androstanol
androstenol