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Welcome to this lecture on the regulation of gene expression by steroid hormones. This
is lecture number 21 in this course on eukaryotic gene expression basics and benefits. On the
last few classes, may be last 5 classes, we have been discussing about how signaling molecules,
which interact with membrane receptors, ultimately activate or repressed transcription of specific
genes. That is how signaling molecules interact in cell surface receptor change or alter gene
expression programs inside nucleus. We have been discussing at least 3 different kinds
of receptors. One of them called GPCR or G protein coupled receptors which contain the
7 transmembrane domains and they what kind of signaling molecules interact with these
receptors and what kind of signaling cascade ultimately leads to activation of gene expression.
We also talked about receptor tyrosine kinases. How growth factors binds to growth factor
receptors, activates the receptors tyrosine kinases and how they through the map kinase
activity lead to ultimately activation or repression of target genes. In the last class,
we discussed about signal transducer and activator of transcription that is how starts or involved
in the regulation of gene expression by cytokines. There are number of other cells of a signaling
molecules and cells surface receptor pathways but we I gave you these 3 different pathways
as an example of how molecules interacting with cell surface receptors can ultimately
transudes signals through into the cell and activation of specific kinases can ultimately
lead to the activation of specific transcription factors. These results in the activation or
repression of gene expression and we also mentioned that there is lot of cross talk
goes on between these different signaling pathways and I gave you some of the examples.
What we will do today? We will now go into the inside the cell and discuss about those
molecules which can diffuse through the cell membrane and enter inside the cytoplasm and
then bind to specific inter cellular receptors and how that results in the activation or
repression of specific target genes. One group of molecules which do this or steroid hormones.
So, what we will discuss in this class is how steroid hormones, what are steroid hormones
and how steroid hormones enter the cell and bind to specific surfaces specific inter cellular
receptors and activate or repress transcription of specific target genes? So, this is what
is the I have shown a schematically here. So far, we have been discussing about molecules
which bind to specific receptors on the cell surface but today we are now going to talk
about molecules which can diffuse through the cell membrane, bind to specific receptors
and often when these molecules bind to these receptors, it results in the conformational
change.
These modified receptors now often dimerize and this dimerize then goes inside the nucleus,
binds to specific promoter sequences and regulates gene expression. So, we are going to use steroid
hormones as example of this group of molecules and ask the question how steroid hormones
are able to do this. That will be crux of today's lecture. What are steroid hormones?
Steroid hormones are steroids that act as hormones. So, steroid hormones can be grouped
into 5 groups based on the receptors through which they bind and these are known as glucocorticoids,
mineralocorticoids, androgens estrogens and progestogens and I just given the structure
of here cortisol is a glucocorticoid which is mainly made in the adrenal gland. Aldosterone
is a mineralocorticoid again made in the adrenal gland. Testosterone is a male sex hormone
which is made in the ***. Estradiol and progesterone are the female sex hormones which
are made in the ovaries.
So, let us now and you can see now one type get tell you the name those are all very important
molecules. They control a number of very important cellular processes and they all have more
or less similar structure as you can see they are derived from the cholesterol. So, let
us now try to understand how what are these molecule and how these molecules bring about
their physiological effects? This is the structure of cortisol I have shown here. They are derived
from cholesterol and this is a glucocorticoid which is synthesized from progesterone in
the zona fasciculate of adrenal cortex. The adrenal gland has 2 major regions called the
cortex and medulla. The glucocorticoids are made in the adrenal cortex whereas the epinephrine
and non-epinephrine which we discussed in the previous class which are the molecules
which interacts cell surface receptor, they are made from the adrenal medulla. Now, cortisol
is involved in stress adaptation, elevation of blood pressure, sodium uptake and numerous
effects of immune system including anti information. Many of the blocks which are available in
the market reduce information or glucocorticoids.
So, glucocorticoids act as major anti-inflammatory agents. All the steroids again, it is a mineralocorticoid.
It is produced from progesterone again in the zona glomerular region of the adrenal
cortex. Again, it is involved in the raising of blood pressure, adjusting regulation of
fluid volume in the blood and also in the sodium ion uptake. So, it plays a very important
role. Progesterone is a female sex hormone and it is produced directly from the pregenenolone
and secreted from the corpus luteum of the ovary. It is responsible for changes associated
with the luteal phase of the menstrual cycle and is involved in the differentiation of
mammary glands, a very important hormone in the case of females. Testosterone is a male
reproductive hormone and it is an androgen. It synthesize in the *** and responsible
for all the secondary *** male *** characteristics. Estradiol is again a female hormone, again
produced from the ovary and is responsible for the female *** characteristics.
So, as you can see these 5 hormones are very important. They have very important biological
functions and they are all synthesized from cholesterol. I have just shown here a slide
that is taken from this particular website which tells you how the glucocorticoids and
the mineralocorticoids namely the cortisol, corticosterone together constitute the glucocorticoids.
Aldosterone and androstenedione they together constitute the mineralocorticoids and how
they are synthesized from cholesterol and what are the various enzymes involved in this
pathway? Ultimately, these molecules are synthesized. We will not go in details of how they are
made but just know that the glucocorticoids and mineralocorticoids, examples cortisol
and aldosterone. They are synthesized by the admiral gland and they are all derived from
cholesterol. Similarly, the gonadal hormones both Estradiol and progesterone as well as
testosterone, again cholesterol is percussive for by synthesis and here is the pathway by
which cholesterol is converted into the testosterone or estradiol. Need not remember the pathway
at this stage but just to tell you that they all are structurally very similar molecules
but they have very different physiological functions and they are produced in different
regions of the body.
These are natural steroid hormone that we talk so far about. They are also what are
called as synthetic steroid hormones again play a very important role, has lot of biomedical
applications. There are variants of structure that you have just seen here and they have
either more potent against or antagonistic action, mostly antagonistic action. The antagonist
also has very important effects. Glucocorticoid synthetic glucocorticoid for example is called
prednisone, dexamethasone, triamcinolone and so on and so forth. Synthetic mineralocorticoid
fludrocortisones these are all derivatives of the basic structure which I have shown
in the previous slides. Many androgens in fact, they have very important role especially
those athletics for example. Athletes who want to have more muscles who want to win
races much faster; they take what are called as anabolic steroids which are actually banned.
Athletes are not supposed to take these anabolic steroids, what is called as a dope test and
if you test positive in this dope test, you cannot participate in many of these competition.
These anabolic steroids are nothing but synthetic steroid hormones, especially synthetic androgens,
oxandrolone, nandrolone and so on and so forth, very important in the sports area. Estrogens
again have a synthetic estrogen, diethylstilbestrol called DES and you have synthetic progestins,
non-ethindrone, medroxyprogesterone acetate so on so forth. So, these steroid hormones
are very important biomedical applications because they are very portent molecules that
alter the physiology. Now, some hormones have been around for a long time. In fact, steroid
hormones have been known to exist since the early twenty century. However, it is only
in the early in 1960s and especially in late 1980s, the molecular mechanism of action of
the steroid hormones really became very clear. So, it was only until, it is not until early
1960s that the idea of specific hormone binding molecules in the target tissue of these hormones
began to emerge. So, till 1960, people did not know how these hormones are working but
then when bio chemistry in early 1960s or 70s, the biochemistry ruled the world in the
biology and when the biochemistry starts grinding the tissues and started looking what kind
of protein molecules bind to this. Steroid hormones, it should become very clear tissues
which are response to this hormone molecules actually contain proteins which bind to this
hormones steroid hormones. So, the concepts that these hormones may be acting by actually
binding to specific receptors began to emerge by bio chemical studies that carry carried
out in the early 60s. The analysis of the steroid hormone receptors
had relied largely on bio chemical techniques as I said the one of the major methods by
which people use to study steroid hormones, the late 1960s and early 1970s is you label,
use a radio label steroid hormone and then you see how the radio label hormone goes and
binds to protein that results and then try to monitor to the kinetics of binding and
then see where this receptor is localize and so on and so forth.
So, primarily bio chemistry contributed a lot to understanding the molecules of the
protein molecules which actually bind to the steroid molecules in the early 60s but it
is only after the genes encoding these receptors were cloned, it became possible to carry out
detailed studies of various functional domains of the receptor. So, the best that bio chemist
could do is to see what kind of molecules bind and what is the affinity of this steroid
hormones to these molecules and with a great difficulty they could actually purify some
of this receptor molecules.
In late 70s some of these proteins or the receptor molecules could also be sequence,
so the partial amino acid sequence of some of these steroid hormone receptors were available
but complete characterization was not possible using this bio chemistry alone but the late
70s and the early 80s started was the drawn of molecular biology. It became to clone genes;
it became to identify the amino acid sequence of proteins using the genes sequence so on
so forth. Once restriction enzyme is cloning, technique became available that made a very
major role in understanding the function structural and function of steroid hormone receptors.
So, what I am going to do is go through take you through a brief historical perspective
of what kind of people are actually contributed a lot to the understanding of the steroid
hormone receptors structure and function and what is the historical perspective of this
filed. That is what we will discuss in next few slides. Now, as I said bio chemistry played
a major role in understanding the structural function of steroid hormone receptors. For
example, in the 1968 some of the studies actually led where you take a tissue, homogenize a
tissue and then do a different gentrification by you can separate nuclei and you can separate
a cytoplasm and then with they took this different faction and see where is the protein which
is able to bind to this steroid hormone is present.
Based on such studies, people actually showed that when there is no hormone, this binding
protein is actually present in the cytoplasmic fraction but the movement we have hormone,
this activity sifted to the nucleus. So, this kind of bio chemistry started giving an idea
of how these molecules actually act or how these molecules are able to bring about physiological
response and they clearly told that these molecules actually enter inside the cell and
binding to specific intracellular proteins. Again, once people started purifying using
these kinds of technical localization studies and again using bio chemical purification
techniques, people have started purifying this receptor protein from some of this tissue.
Once you got a purified receptor, you could actually do a partial styptic digestion of
this receptor or find out N terminal acid sequences or internal peptide sequencing and
based on these peptides, as well as the purified receptor, you can immunize either mice or
rabbits and you can generate antibodies. So, once you have antibodies which are specific
for this kinds of steroid hormone receptors, then people use this antibodies to ask the
question where exactly this receptor are present. For example, if you have a specific tissue
which has a binding protein for that particular steroid hormone, you do what is called immuno-fortis
technique where you order receptor anti-antibody to specific that particular receptor. Then
we have what is called the second antibody which is always conjugated to florescent molecule,
it could be what has called florescent isothicyanate or and so many other modern dyes which under
florescent microscope either give a blue colour, green colour or a red colour.
So, using this kind of immuno-fluorescence technique people and using receptor specific
antibodies, people could actually demonstrate that these receptors when there is hormone
inside the cell they are actually present inside the nucleus. So, picture began to emerge
that these steroid hormones basically act by binding receptor molecules present inside
the cytoplasm and once the hormone binds the receptor, this receptor is going to the nucleus
and probably it is activating the transcription of specific genes.
That is how these steroid hormones are able to bring out specific biological effects.
This is the paradigm that began to emerge, so many of this bio chemical studies that
was carried out in the late 1960s. So, this is what I have summarized. What I just told
you here, it is generally thought that unoccupied steroid receptors can exist in the cytoplasm
while the occupied receptors act in the nucleus on specific target DNA sequences but these
are all again not much experimental evidences available, it is mostly conjunctional. So,
when bound to the hormones cytoplasm, hormone receptors move to the nucleus and the purified
receptors probably are going and binding to specific DNA sequences. Some of the key experiments
which were done again using bio chemistry and little bit of molecular biology because
as I said the late 70s and 1980s is the one that was the don of molecular biology.
A number of molecular biology techniques became available and people started using, especially
bio chemist who were actually doing protein purification and protein characterize and
technique now realize that they could answer many of this question using many of these
molecular biology techniques by looking at the transcription by looking at the genes
and so on and so forth. So, what for example Keith Yamamoto's group actually did in 1983.
They published paper in cell in 1983, sequence specific binding of glucocorticoid receptors
in MTVDNA at sites within and upstream of the transcribed region. So, that is virus
called mouse mammary tumor virus and this virus response very well.
The trans for the transcription of this virus, you require glucocorticoids. So, people like
Yamamoto started asking question, how does this mouse mammary tumor virus promoter is
getting activated by glucocorticoids because glucocorticoids is producing in our body and
here is a mammalian virus and the transcription of this virus is actually getting activated
by a hormone which is produced by our body. So, what they did? They actually took what
is called the long terminal region long terminal repeat or LTA of these particular MMTB virus
or MTV virus and then ask the question where exactly does the glucocorticoid receptor binds
because by this time, it is very clear. All the effects of glucocorticoids are probably
modulated to the glucocorticoid receptor. So, if the glucocorticoid is activating the
expression of this mouse mammary tumor virus, people ask the question. The glucocorticoid
receptor is must be doing something to the virus and the first question they ask this
does it go and bind to specific DNA sequences within the mouse mammary virus tumor DNA.
In fact, actually showed that there are about 5 regions within the MTVDNA, they are specifically
bound by purified glucocorticoid receptor and one result upstream of the transcription
start site and others are distributed within the transcribed sequences between 4 to 8KB
from the initiation site. So, for the first time studies like this are actually shown
that here is a receptor mammalian receptor that is actually binding to some specific
sequences within this viral genome and this binding is probably responsible for activation
of expression of the viral genes.
So, such studies with purified steroid hormone receptors demonstrated that they are likely
to be sequence specific DNA binding proteins and they are likely to be transcription factors.
So, this is what emerged for many of these bio chemical studies. So, classical purificate,
classical ligand, receptor interaction studies, understanding their kinetics of binding and
affinity of this ligands to this receptors as well as bio chemical purification techniques
followed by several localization using immuno-fluorescence as well as and more late 70s and early 80s
using some of the molecular biology techniques, it became very clear that one of the major
mechanisms by which these steroid hormones are acting is by binding to the steroid hormone
receptors and these steroid hormone receptors are then going inside the nucleus and bind
to specific regions within the DNA sequences. That is how they are able to bring out specific
physiological effects. So, this is the paradigm that emerged before the molecular biology
era started.
The most important phase in the study and understanding of the steroid hormone receptor
function began with the cloning of steroid hormone receptors. So, I am going to give
you a brief historical prospective of how this steroid hormone receptors were cloned.
Now, studies from a number of laboratories such as those headed by Elwood Jensen, Bert
O'Malley, Keith Yamamoto and many others, I am not going to quote everybody who worked
in those areas these are some of the very important people who made very important contributions
to the understanding of steroid hormone receptor functions.
All these studies actually led to the hypothesis that activated steroid hormone receptors go
and bind to specific DNA sequences in the nuclear of the target cells and this is what
induces the transcription of these genes leading to specific production of specific proteins.
So, all the bio chemicals studies and immuno-localization studies and typical receptor ligand interaction
studies, all these things pointed out that when you add a hormone to this steroid hormone
to the cell, it is entering the cell, binding to this inter cellular receptors. Then it
goes inside the nucleus and this binding of the receptor to specific DNA sequences results
in the synthesis of specific proteins and that is how the steroid hormones are bringing
about their physiological responses.
Now, by the end of 1980s when techniques for cDNA library screening and cloning and sequencing
DNAs became a routine, the first receptors cDNA encoding the glucocorticoid receptor
was cloned by Ronald and Evans in the Salk Institute in La Jolla California united states
of America. So, 1980s as I told bond was the golden era of biology because the molecular
biology techniques became available. People realized that you can actually purify messenger
harness for a specific protein, encoding this specific protein and you can derive a cDNA
of those and you can actually clone those DNAs. So, from millions and millions hundreds
and thousands of genes which are available, you can actually pull out a specific gene
or specific cDNA coding for a particular protein. So, once the creation of cDNA libraries and
how to screen these cDNA libraries and how to isolate the specific cDNA became available,
people began to understand can we clone a cDNA that clones for or can we clone messenger
RNA that actually syntheses this steroid hormone receptors. The first one was done by Ron Evans
Salk institute in the late 90s the early 1980s. Once they receptors glucocorticoid receptor
was cloned, this was soon followed by cloning of the cDNA encoding for estrogen, progesterone,
androgen so on and so forth cloned in subsequent years. So, I am going to spend some to tell
you how actually all these things were done because these are land mark papers in the
area of steroid hormone receptor structure and function.
So, let us now see what exactly they did. So, the first paper on the characterization
of the cDNA encoding the glucocorticoid receptor was published in journal nature in 1985-1986
by Ronald Evans Salk Institute and his entirely primary structure and expression of a functional
Glucocorticoid receptors cDNA. These how would considered as a land mark paper in the area
of steroid hormone receptors structure and function because this marked the beginning
of a new era in the bio chemistry and this paved way are this probably is primarily responsible
for our current understanding of the steroid hormone receptors structural function. So,
what this group actually did is to demonstrate that there are 2 forms of glucocorticoid receptors
cDNA's. So, what basically they did? They took the RNA isolating from a tissue which
express a glucocorticoid receptor, converted them into cDNA by using reverse transcription
and so on and then took the cDNA's of all these messengers or sensor in the tissue and
then put them in the page vector and then made a library.
So, I have basically made a cDNA library and then using either antibody specific for glucocorticoid
receptor or using oligonucleotides which specifically or digging the oligonucleotides that corresponds
to the specific amino acid sequences of this glucocorticoid receptor which is available
at that time because the purified receptor could be cleaved and the partial amino acids
sequence could be identified for some of the peptides using either oligonucleotides that
corresponds to those peptide sequence or by using antibodies rising purified glucocorticoid
receptor. You basically, screen these libraries and ask the question which phase clone are
for the cDNA that actually cores for the glucocorticoid receptor.
So, by cDNA screening, cDNA library screening they have actually identified 2 clones and
one of them cloned for a glucocorticoid receptor of 777 amino acid and they called it as glucocorticoid
receptor alpha. Another cloned for a 742 amino acid protein which they called as a glucocorticoid
receptor beta and you can see this is the correct knowledge of glucocorticoid receptor
structure and the foundation for this was actually laid in the year 1985-86 by Ronald
Evans group. We now know that all these amino acids are encoded from about 9 hexons. This
is the hallmark from 1 to 9 here and today, we know that the glucocorticoid receptor structures
actually consists of what is called as a N terminal domain and it consists of a DNA binding
domain which actually comes from hexon 3 and 4 and consists of what is called as ligand
binding domain to which the glucocorticoid goes and binds. So, the alpha is after this
whereas the beta is truncated here.
Now, in the same year from the same group, it also became another important land mark.
Paper was published where it was reported that the domain structure of the glucocorticoid
receptor and its relationship to the v-erb-A gene and its relation to v-erb-A oncogene
product. Now, I can see here the importance of molecular biology.
Now, the bio chemical purification studies could only at the most partial amino acid
sequence, all they could raise antibodies but nobody could obtain the full length amino
acid sequence of glucocorticoid receptor using conventional bio chemical techniques because
it is impossible for you to sequence the entire. How many amino acids? Almost 777 amino acids,
you cannot do typical amino acids sequence of these proteins this 2 v-erb recurring a
draw but once the cDNA sequences are available from the cDNA sequence; you can actually deduce
the amino acid sequence and based on the amino acids sequence, people ask the question what
kind of a protein is this? What kind of functional domains are actually present in this glucocorticoid
receptor? Based on such studies in the subsequent paper they actually shown that this glucocorticoid
receptor some of the domains actually resemble to that of an alkaline protein called v-erb-A.
Now, what is this v-erb-A? The v-erb-A is an oncogene product of a virus called avian
erythroblastosis virus or AEV. So, these are viruses which when we infect, it actually
causes cancer and one of the major proteins which is responsible for causing cancer in
this virus is a protein called v-erb-A. What the studies of this group actually shown is
that the structure of the glucocorticoid receptor very much resembles the structure of this
oncoprotein. So, you can see people who have been studying steroid hormone, suddenly realize
that there is some relationship between steroid hormone receptors and cancer because here
is a viral protein which is causing cancer and the structure of protein is resembles
very much that that of that glucocorticoid receptor. So, people asked the question, what
is the link? So, they predicted in this paper that the oncogencity of this virus may result
in part from the inappropriate activity of a truncated steroid receptor or a regulatory
molecule encoded by v-erb-A. So, the movement they found that structurally
these two proteins are related. People realised that the mechanism by which this oncoprotein
is acting causing cancer, may be similar may be because of inappropriate signaling. So,
they suggested that a mechanism by which transcription factors may facilitate transformation. So,
these kinds of studies clearly showed that transcription factors may be involved in similar
transformation leading to cancer and they also identified another short region in the
human glucocorticoid receptor which had very high degree of homology to certain proteins
which are involved in the regulation of development like The Drosophila homeotic proteins encoded
by Antennapedia and fushi tarazu etcetera.
Now, we will come back to this transcription factor regulation in development sequence
places but what I am trying to tell you is that once the cDNA for the glucocorticoid
receptor was cloned and the domains structure was analyzed, it became very clear that this
is like to be a transcription factor. There is something look like a DNA binding domain
and it also had a homology to very important viral protein which causes cancer and people
realized that the mechanism by which this virus are causing cancer may be because of
inappropriate activation of some of the genes which are actually activated by steroid hormone
receptors. Now, they soon realized that the subsequent here 2 groups are actually published
a paper where they actually demonstrate the v-erb-A gene is nothing but a thyroid hormone
receptor. So, the link between cancer and hormones became
firmly established when they realized that one of the viral oncoprotein which very much
resembles that of a glucocorticoid receptor actually course for a thyroid hormone receptors.
I can see as I have just highlighted some of the important findings from this paper
where this group, again from Ronald Evans lab actually showed the c-erb-A gene encodes
a thyroid hormone receptor. What they showed is that the cDNA sequence of homology and
c-erb human the c-erb-A the cellular counterpart of the viral oncogene, v-erb-A indicates that
the protein encoded by the gene is related to steroid hormone receptors and binding studies
with the protein show it to be a receptor for thyroid hormone.
So, they took the gene encoding for the thyroid hormone receptor, did what is called as invitro
transcription and translation and that is you take the RNA, translate in the invitro
and this invitro translate protein was able to bind to thyroid hormone. So, here is a
viral oncoprotein involved in cancer that seems to thyroid hormone clearly saying that
there is a link between the hormone signaling and cancer. Again, here in the same you can
see these two papers are published in the same issue of nature back to back. This is
from Vennstorm lab in Europe where again it is showed that hormone binding and localization
of the c-erb-A protein suggest that it is a receptor for thyroid hormone, a nuclear
protein that binds to DNA and activates the transcription.
So, the product of viral oncogene is defective in binding to the hormone but still localised
to the nucleus. So, you can see here is a viral oncoprotein which binds to a DNA but
it is not bind to thyroid hormone clearly indicating that if we have receptors which
have defective in ligand binding or which are hormones binding and such receptors may
cause inappropriate signal transition pathways activate inappropriate oncogenes. This is
what can lead to ultimately cancer.
So, these kinds of studies established a link between viral virus induced cancers or a viral
oncogene products and hormone signaling pathways. So, the cloning of glucocorticoids receptors
are in the same time Ron Evans group clones glucocorticoid receptor. A number of other
laboratory started cloning other receptors because they all had either antibodies of
specific receptors or they had the partial peptides sequence amino acids sequence or
salten peptides of this various receptors. Pears Chambo group in France for example took
the antibodies against the glucocorticoid estrogen receptor as well as they made ologo
neutralize against peptide amino acid of specific peptides of the estrogen receptor and they
cloned the estrogen receptors cDNA. So, they reported the cloning of the cDNA of estrogen
receptors in the breast cancer cell line MCF-7 and expression of the ER cDNA in hela cells
produces protein that has a same relative molecular mass and binds oestradiol with the
same affinity as MCF-7 ER. So, it became very clear that protein which
is actually produced from this cloned gene is more over the same as the estrogen receptor
that is expressed in a well-known breast cancer cell line. So, this they again they also reported
that there is extensive homology between the estrogen receptor and the erb A erb-A protein
of the oncogenic avian erythrocytosis virus. So, all these became clear that whether you
clone glucocorticoid receptor, whether it is an estrogen receptor, they all seem to
have similar structure and the structure is very similar to that of an oncoprotein encoded
by oncovirus or a tumor virus.
So, all these studies clearly led to the proposal or hypothesis that there exists a super family
of oncogenic hormone receptors. So, there are certain oncoproteins which are highly
homologous to the hormone receptors and this super family of receptors may be actually
involved in viral transformation. So, both Chambon as well as Roan Evan group, actually
proposed that these human steroid receptors as well as some of this viral oncoproteins
like erb-A may actually constitute a super family of enhancer binding proteins and they
may actually cause cancer. Once the glucocorticoid receptor cDNA and estrogen receptor cDNA was
cloned, subsequently number of other steroid hormone receptors was cloned. For example,
within 1960s 1968 for example the chicken progesterone receptors cDNA was cloned as
well as the human androgen receptor cDNA was cloned.
So, once all these cDNA for this steroid hormone receptors was cloned it became very clear
that they all encoded super family of hormone receptors. This I am just putting a slide
which I have already explained here. What was the strategy that was employed for cloning
of all these genes including steroid hormone receptors? The strategy is to make a cDNA
library that is you isolate messenger RNA from all these tissues which are expressing
these specific receptors and then you convert all this messenger RNA into cDNA and then
you clone this cDNA into phage vector and make a phage library or a phage DNA library.
Then take this phage cDNA library and you either probe them with antibodies against
specific receptors which have been purified from various cell types. You take these antibodies
against the purified receptors or make oligonucleotides degenerate oligonucleotides corresponding
to the amino acid sequence of specific receptors and using either any labeled oligonucleotides
or use antibodies, you can screen this phage libraries phage reading libraries. That is
what they can isolate this cDNA clones and then you sequence this cDNA clone and reduce
an amino acid from cDNA sequence and see what kind of a protein that they are coding for.
So, this was a strategy that was used for isolating the various steroid hormone receptors
in the early 80s and late 1980s. So, once all these studies were done, you have the
receptor of glucocorticoid receptor, you had the receptor of estrogen receptor, thyroid
hormone receptor so on and so forth. It became clear from all these studies that all these
receptors had some common structural domain. For example, all these receptors have what
is called as the ligand binding domain which probably binds the hormone. It is called as
the hormone binding domain or the ligand binding domain. The receptors also has what is called
as DNA binding domain in the middle and this probably responsive for binding specific sequences
and it also what is called as amino terminal hyper variable region which was not that will
consult between various receptors that then go so on and so forth. So, some of the three
major functional domains of this receptors were a ligand binding domain, the DNA binding
domain and hyper variable amino terminal domain. So, this is for the major domains which are
present in all these receptors.
Today, we now know that there are much more detailed information available on the various
domains of the steroid hormone receptors. For example, we now know that this amino terminal
region which is actually shown as A slash B actually contains what is called as an activation
function 1. This is actually responsible for ligand independent activation of steroid hormone
receptors and we also know that the region known as C, actually course for a DNA binding
domain and this DNA binding domains consist of 2 zinc fingers.
We have discussed about zinc fingers in the previous classes when we talk about the structure
and function of various DNA binding proteins and this steroid hormone receptors contains
2C2H2 type of zinc fingers and we will look at this structure in a few more minutes. It
also be very clear people started to looking at the mechanism by which this DNA binding
domain acts while discuss in more detail in a few more minutes and this DNA binding domain
what is called as the P box and what is called as a dimerisation region and plays a very
important role in DNA binding function of the steroid hormone receptor.
As I said all these steroid hormone receptors are present in the cytoplasm when there is
no hormone, they go into the nucleus in the presence of hormone. So, they contain what
has called as nuclear localization signal and this nuclear localization signal is massed
when there is no hormone once the hormone binds its induced conformational change. So,
the nuclear localization signal gets exposed. Therefore, receptor goes inside the nucleus.
Now, as was ligand binding domain, the ligand binding domain has two important functions.
It has a ligand dependent activation function that means there is an activation function
2, whereas the one in the amino epidemic terminal referred as activation function 1. When ligand
binds, this activation function is activated and now this in the presence of ligand, the
receptors able to interact with the general transcription machinery and the RNA polymerize
resulting in the activation of transcription. The domain also in the absence of ligand in
certain receptor can act as a repressor. So, the cloning what of this cDNA's of these receptors
paved way for a very detailed understanding of the various functional domains of these
various steroid hormone receptors.
Now, once all these receptors were cloned, there also many it also became clear that
the structure of this steroid hormone receptors made homology to other non-steroid receptors,
for example thyroid hormone receptors. Thyroid hormone is not a steroid hormone but receptor
for thyroid hormone was very homologous to then of the glucocorticoid receptor or estrogen
receptor. So, people soon realized that there exists a nuclear receptor super family and
this nuclear receptor super family contains not only steroid hormone receptors but also
contains other non-steroid hormone receptors or other hormone receptors and which are not
steroids. So, all these based on the structural homology were grouped under one nuclear receptor
super family. So, by sequencing cDNA's of various hormone receptors and deducing the
amino acids sequences, all this were grouped under a single nuclear receptor super family.
Two broad categories of this receptors exists in this one are called as type 1 receptors,
another called as type 2 receptors. The type 1 receptors, basically consists of steroid
hormone receptors. They undergo nuclear translocation of the ligand activation and bind as homodimers
to inverted repeat DNA half sites refer to as the hormone response elements. So, the
DNA sequences to which the steroid hormone receptor binds or HRE or Hormone Response
Elements. Glucocorticoid well bind to glucocorticoid response element, estrogen will binds to estrogen
response elements, thyroid hormone bind to thyroid hormone response element so on and
so forth. So, in general they are known as hormone response elements.
So, the example for the type 1 receptor are receptors activated by steroid ligands such
as glucocorticoid receptor, mineralocorticoid receptor, estrogen receptor, progesterone
receptor and androgen receptor. So, all these steroid hormone receptors are classified as
type 1 receptors because usually they stay in cytoplasm in the absence of the hormone
and once the hormone binds the conformational change, then going to the nucleus and often
bind what is called as inverted repeated sequences. We will come to that in a minute whereas the
type 2 receptors, they often are retained in the target cell nucleus regardless of the
presence of the ligand. So, unlike classic hormone ligand receptors which are present
in cytoplasm in a hormone and goes into the nucleus only when there you have hormone.
The type 2 receptors are already present into the nucleus, irrespective of whether the hormone
is present in the hormone or not. So, they are nuclear receptors. Whereas, steroid hormone
receptor cytosolic in nature in the absence of hormone and nuclear in nature in the presence
of hormone and the type 2 receptor is always nuclear in the nature The function of ligand
is actually to unfold or to activate the transcription activation function of the type 2 receptor.
So, in the absence of hormone, although the receptor is bound to the DNA, it does not
activate transcription but both the hormones binds; it induces a conformation change. Therefore,
the receptor can activate transcription. Examples for type 2 receptors are thyroid hormone,
retinoid acid, vitamin D so on and so forth.
We will discuss type 2 receptors in the next class but today we will confine ourselves
to only for the type 1 receptors which basically comprise of the steroid hormone receptors.
Now, so it became very clear that these steroid hormone receptors act or bring about their
physiological effects like primarily binding to what are called as the hormone response
elements which are present on the promoter regions of various genes. So, once the hormone
binds, the intercellular receptors now functions as transcription factors. They go and bind
to the hormone response elements in the promoter regions of various genes and this is how they
act as transcription of various genes. So, all the genes which are the target called
glucocorticoid hormone. They invariably contain a hormone response element in the promoter
region and that is how transcription of these genes are getting activated in presence of
those hormones.
So, if the target gene needs to be activated by glucocorticoid, then you should contain
glucocorticoid response element. If the product gene is to be activated by estrogen, it should
contain an estrogen response elements and so on so forth. So, by having specific hormone
response elements in the promoters of these target genes, these hormones by through their
receptors are able to bind this in the sequences and bring about transcription activation.
So, this became the general mechanism by which steroid hormones bring about transcription
activation. Now, one of the important experimental techniques
that actually played a way for understanding the structural function of this nuclear receptors
and the steroid hormone receptor is assay called as cis-trans cotransfection assay.
It is because of this assay, it became possible to identify the various functional domains
of these steroid hormone receptors. I have already discussed this cis-trans cotransfection
assay in one of the earlier classes, especially the introductory classes of this course but
I do not mind repeating again. Basically, what this actually means is that this is a
transient transfection based hormone response element reporter assay developed again in
the laboratory of Ronald Evans at salt institute and it became the tool of choice for investigating
the mechanism by which nuclear receptors regulate their target of genes.
So, this is a very important assay and because of this assay that people could identify the
various functional domains steroid hormone receptors. What is this in this assay? A cDNA
encoding the receptor or part of the receptor is transfected into a suitable cell line along
with a reporter gene usually can be luciferase gene link to a promoter controlled by one
or more of the hormone response element specific for the receptor being studied. So, basically
what to do in this assay is that you take the cDNA that codes for, let me say I want
to see whether glucocorticoid receptor actually activated from a promoter or not or whether
a promoter actually contains a glucocorticoid response element or not.
So, what I do I take the promoter sequence or I take the sequence which suspected in
the glucocorticoid response element and link it to a luciferase gene which is a reporter
gene, it can also be or can be transferase, there are number of such reporter genes. So,
take the luciferase gene, put it down stream of this suspected promoter which is response
glucocorticoid and put this plasmin inside the cell. This is called as the cis factor.
Now, you take another plasmin which contains let us say for example a well-known viral
LTR viral promoter which is known to be expressed by host transcription factor in a number of
cell line. So, clone the receptor cell for glucocorticoid receptor under this promoter,
so that when you put this plasmin inside the cell, the transcription factors present inside
the cell go and bind to the promoter and they express the receptor. Once the receptor is
expressed, the receptor will now go on and bind to the cis vector, the promoter region
of cis vector and now if we add ligand, it will activate the transcription. So, if the
cis plasmin contains the sequence for binding of the hormone receptor, then when the receptor
is expressed from transplasmade, it will go and bind to luciferase vector and induce the
express the luciferase gene. You can then you can measure in the luciferase activity
and then you can now demonstrate that yes, this receptor can actually activate transcription
activation from a specific region. So, by using this assay not only you can identify
hormone response elements in promoter region of any gene, you can also bisect out what
region of the receptor is important. For example, you can make mutations by DNA binding domain
and ask the question can it go and bind to DNA? Can it now activate transcription or
you can change amino acid sequences within the DNA binding question and ask the question
how is this specific domain or you can switch the ligand binding domain. For example, since
I have DNA for estrogen receptor, have a cDNA glucocorticoid receptor, I can take the ligand
binding domain of the glucocorticoid receptor and fuse it to estrogen receptor. Now, if
I take the primary receptor and put inside the cell and since the glucocorticoid receptor,
now has an estrogen receptor cDNA, now it will activate transcription from glucocorticoid
response element in response to estrogen but not in response to glucocorticoid. These kinds
of experiment done using this assay clearly indicate that these steroid hormone receptors
have a modulus structure.
You can take the DNA binding group of glucocorticoid receptor and put it on estrogen receptor or
you can take then DNA binding of estrogen receptor, put it inside glucocorticoid repeat
and the deal is they start activating from each other response element that is the glucocorticoid
response receptor contains estrogen response receptor DNA binding domain will now start
activating transcription in estrogen response element or you can do other way also. You
can take ligand glucocorticoid receptor binding domain or the ligand binding domain glucocorticoid
receptor and switch it to that of the estrogen receptor. Now, they start activating transcription
with each other ligand. So, these kinds of cis cotransfection assays, they would say
for characterization of the DNA binding domain as well as ligand binding domain of the various
steroid hormone receptors. So, such studies actually demonstrated that
steroid hormone receptors actually bind to hormone response elements and this actually
led to the characterization of the various steroid hormone response elements. For example,
hormone such as progesterone, androgen, glucocorticoid and mineralocorticoids, they all seem to bind
to a specific the same kind of response element containing AGAACA separate and TGTTCT separated
by three nucleotides. This is actually called as an inverted repeat. You can see AGAACA
and the opposite stands as TGTTCT, so AGAACA and the opposite stand also AGAACA and therefore
is called inverted repeat. So, AGAACATGTTCT and the opposite stand again lead as AGAACA,
so how this same AGAACA sequence on the two sides of the double DNA. So, these are called
as palindromes or inverted repeat sequences. So, many of these steroid hormone receptors
bind to the repeat sequences, it should become one of the hallmarks of hormone response element.
Interestingly, all these four receptors progesterone, androgen receptor, glucocorticoid, mineralocorticoid,
they all bind to a similar sequence which consists of what are called as two half sides.
The AGAACA is one half side, TGTTCT is another half side and these two half sides are separated
by three nucleotides, it can be any nucleotides. So, when you have this kind of sequence in
the promoter region of gene, you can say this is lightly to be activated by any one of these
hormones. Similarly, if you take estrogen receptor, estrogen receptor actually binds
to a sequence called AGGTCA, 3 nucleotides TGACCT. So, you can see the only difference
between estrogen receptor or estrogen response element and a glucocorticoid response element
is two nucleotides. So, if the sequence is AGAACA, it becomes glucocorticoid element
instead of AGAACA. If it is AGGTCA, it becomes estrogen response element. So, you can see
the physiological effects of estrogen is quite different from physiological effects of glucocorticoids
but if you see at the molecular level, the only these two of the ways very fine difference
that distinguishes between the estrogen receptors and progesterone receptor target genes.
So, they target gene promoter target genes, the promoter contains AGAACATGTTCT becomes
responsible to glucocorticoid. If the same sequence instead of the 2A replaces such by
AGT, it now become a response for estrogen receptors. So, this is a very fine difference
exists at the molecular level between genes that are responsible for glucocorticoid and
the genes that are responsible to estrogen and very fantastic experiments that actually
done at the future characterized DNA binding through steroid hormone receptors. So, it
became clear that these all these steroid hormone receptors whether it is GRMRPRE RRAR,
they all contain two zinc finger domains and this zinc is coordinated by cystine-rich.
So, they contain the C4 type of zinc fingers and it has been very well shown that the DNA
binding domain actually contains two very important regions called as P box and D box.
The D box refers to dimerise box or a distil box and P box is the proximal box and many
number of people actually could do mutation within this the DNA binding domain and ask
the question, what are the amino acids residues which are important for DNA binding. For example,
if you now mutate this particular A adenine in this DNA binding alinine in this region.
If you mutate that a quanine, now this receptor can no longer dimerize. So, if you mutate
the IM residue that of in this amino acid position 458 in the glucocorticoid receptor
to the glucocorticoid receptor cannot dimerize. So, can now just go back and tell you here,
since the hormone response amino consists of palindromes that means you have two half
sides AGAACA1 on one stand and AGAC on other stand. Therefore, the steroid hormone receptors
in order to activate transcription and they go on and bind as dimers.
So, one hormone recognizes its one half sides on one stand and the other hormone recognizes
on the other stand. In order to follow to bring together, we need a dimerisation domain.
There is a dimerisation interface domain and there is also dimerisation interface in the
ligand binding domain of all these steroid hormone receptors. So, using this kind of
cis-trans cotransfection assay people have to identify what are the important amino acid
residues that play very important role in DNA binding. Like I said in the case of glucocorticoid
receptor, one mutation actually can abolish the dimerisation function of the receptor.
A very important experiment that was done in the 1989 in the year 90 by the two groups,
one headed by Gordon Ringold lab as well as Ron Evans lab in the Salk Institute, they
actually demonstrated by making specific mutation within this DNA binding domain, you can actually
change this specificity of one receptor to other.
For example, in this paper published in cell where they actually showed two amino acids
within the knuckle of the first zinc finger specify DNA response element activation by
the glucocorticoid receptor. What they actually showed in this paper is that the ability of
glucocorticoid and estrogen receptors were discriminated between the close related response
element results in two amino acids located between the two cysteines in the C-terminal
half of the first finger.
I have told you glucocorticoid response element is AGAAC half side whereas the estrogen receptor
instead of AGAACA it is AGGTCA. So, only two nucleotides are different. What is paper actually
include is that if you change two amino acids in the DNA binding domain of the glucocorticoid
receptor is that to recognize that glucocorticoid receptor is now recognized to that receptor.
A similar paper was published in the same issue of cell and this is the amino acid by
again from the Ron Evans group. Again the same issue of cell where actually showed as
that simply change in rising residue to glutamate in the base of the first zinc finger in the
P box, you can now convert glucocorticoid receptor to that of estrogen receptor with
respect to DNA binding specific is concerned.
So, if we simply mutate one amino acid glazing to glutamate instead of this receptor recognize
glucocorticoid response element, it will go and recognize estrogen response element. So,
you can see we have a number of steroid hormone receptor from the gross structure is the same.
They all contain a zinc finger domain but minute amino acid differences between these
receptors actually distinguish whether this was going to bind glucocorticoid response
elements or whether go and bind to a estrogen response element. So, palindromic and all
the steroid hormone response elements exists as palindromic sequences and the receptor
actually goes and binds as a dimer as shown here. One hormoner comes and binds one half
side and another hormoner comes and binds another half side. That is how there is a
bind dimer to the target DNA sequence and activates transcription and as I mentioned
here the glucocorticoid response element and estrogen response element differ from each
other from two nucleotides if is AGAACA half side is a glucocorticoid response element
if the AGGTCA half side it becomes estrogen response element. If you just make one amino
acid mutation within a DNA binding domain of glucocorticoid receptor instead of recognizing
this sequence, now the glucocorticoid receptor recognize this sequence.
So, you can see how beautifully the nature has evolved target genes specific of this
steroid hormone receptors. Now, one can ask question, you have glucocorticoid receptor,
mineralocorticoid receptor and androgen receptor, they are all recognised the same sequences,
then how is this specifically brought about because the mineralocorticoid physiological
effects are different and glucocorticoid physiological effects are different. Androgen receptor physiological
effects are different but they all are binding the same sequence.
So, how is that specific stage brought about? How specific physiological response are brought
about by these different receptor, despite the fact that DNA binding by same sequence.
So, the answer is although many receptors like GR, MR and AR, they all recognize with
the same sequence, the specificity achieved by what kind of receptors actually present.
For example, a tissue which is responsible to glucocorticoids may only have glucocorticoid
tissue which is responsible. Glucocorticoids may have only glucocorticoid. It may not have
a mineralocorticoid receptor, therefore it become responsible for glucocorticoid receptor.
So, by expressing specific receptors in this target types, they can achieve specific target
gene specific receptor. So, very rarely you will find both the PR and GR may be expressed
in the same type of cells. Even they are expressed in the same types of cells; the co-activators
may require for the activation may be present in one receptor but not the other. We discuss
this later in the lectures series.
So, although the target genes specifically may be similar depending up on the cell type
and tissue specific expression of the receptors as well as the different coordinated requirements
like different gene may be activated and different receptor may be activated in different cells.
So, these kinds of experiments have been actually done again by very nice experiment where you
can actually demonstrate this kind of specificities by actually, for example in the estrogen receptor,
how do they demonstrate the receptor can actually activate from the estrogen response element
but not from glucocorticoid response element in the same cell type. You can actually show
you can take the estrogen receptor cDNA and transfer this in an expression that and transfer
this expression into the cells of your interest when the glucocorticoid estrogen receptor
is expressed. Now, if we had an estrogen hormone into this cell, it will now activate only
those genes which can estrogens response element but it will not activate transcription of
those genes containing a glucocorticoid response element. Indicating that these receptors binding
into very specific hormone response elements, that is why they bring out transcription activation.
So, the identification of glucocorticoid receptors and disoffering the fact that this hormone
go and binds to the receptor and then the receptor dimerize and they go and bind to
specific hormone response element. The cloning of the glucocorticoid receptor cDNA as well
as estrogen DNA are one of the hallmarks or land marks in the area of steroid hormone
receptor signal transaction pathways. So, this marked a very important beginning in
the understanding. How this hormone functions? This actually
led foundation for understanding the functions of many other receptors which we will discuss
in the next few classes. Now, what we discussed today is just about one class of receptor,
what are called as type 1 receptors and the estrogen receptors, glucocorticoid receptors,
mineralocorticoid receptor, progesterone and androgen receptor. Now, there is no way huge
family which is called as a nuclear receptor super family and the cloning of these cDNA's
and demonstration that all this receptors actually have similar domain structure and
then people started using the zinc finger cDNA binding domains of the receptors has
proofs and using this proofs, they started screening cDNA libraries of various tissues.
They found that this kind of a domain structure is present number of other receptors and this
led to what is known as a nuclear receptor super family and is a new nomenclature. This
nuclear receptor family includes not only the steroid hormone receptors; it also includes
thyroid hormone receptor, retinoid acid receptor, what is called as a PPAR and so on and so
forth. In the next few classes, we will discuss some of these receptors and ask some question
what is their importance and how do they activate transcription. So, the characterization of
the cloning of the steroid hormone receptors is one of the very important land marks in
the area of the nuclear receptor biology that actually paved way for the characterization
of a number of other receptors which is mentioned here. So, nuclear receptor super family consists
of not only the steroid hormone receptors marked in red but also number of other receptors.
What is very interesting is that many of these receptors actually we know what kind of ligands
bind but there are many other receptors; we do not know what kind of ligands they actually
bind. For example, based on this the nuclear receptor can actually be classified into three
different classes. One is the class 1 receptors which we have already discussed named steroid
hormone receptors which actually bind to palindromic sequences or inverted repeat sequences and
they are usually present in cytoplasm in the hormone and they are going to the nucleus
in the hormone. Tomorrow, in next class we are going to discuss
the class 2 receptors which contain the receptor for a vitamin D thyroid hormones and retinoid
acid and ask the question. How understanding glucocorticoid receptor function has paved
way for the function of this receptors and what kind of sequences these receptors bind.
They are also called what is called as orphan receptor that is these receptors share the
same homology with that of the other receptors. They also contain zinc. They are also zinc
finger transcription factors but we do not know what the ligands for these receptors
are. Some of them actually functions of ligand independent transcription factors and they
also bind to the different sequences.
So, what I have tried to mention in this class is to demonstrate the discovery of steroid
hormone receptors and how the discovery of steroid hormone receptor paved way for discovery
of the other members of the nuclear super family. What I have shown in this slide, these
three important people who actually made a wonderful contribution for this steroid hormone
receptor biology. One is Pierre Chambon in France, Ronald Evans in Salk Institute, California
and Elwood Jenson who actually made very pioneering work before cloning of the receptors actually
began and recognizing their important contribution to this field they were actually awarded called
the Albert Lasker basic medical research award. This often refers as the American noble prize
and it is many people working in this era believe that these people actually may deserve
or actually deserve noble prize and may be awarded noble prize in very near in the future
because they made very important contribution for the discovery of the super family of nuclear
hormone receptors and how this nuclear hormone receptors important for a number of physiological
processes.
I had a privilege of working in Ron Evan lab and so I was working in this lab for three
years and what we worked is about what is called cross coupling of nuclear receptors
with others in the transcription factors and we will discuss that later in this course.
So, what are the people who made a very important contribution had a privilege of working with
him. I just listed some of the key references which are listed here which made a very important
what are called land mark papers, how receptors were cloned and how they were identified and
if you go through some of this land mark papers, you will understand and appreciate some of
the efforts that has gone into this discovery of steroid hormone signaling. I think I will
stop here.