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Actin filaments : Formin :
Poles: microtubules
Wires: Actin filaments
Microtubules: 13 proto-filaments in total. Initially in plant cells, but then in animal cells.
Plant cells and animal cells have basic organelle structure.
Why do Cells need microtubules? Very tiny. Persistence length is one millimeter. Microtuibule organizing center, the centrosome.
Cilia are bundles of microtubules. They push things out of the lungs. Smoking would cause cilia to integrate. Severe coughing is
caused by cilia regrowth in the lungs.
Flagella: sperm has flagella. clamidemoses in sperm contains the flagella. 9 doublets surrounds 2 singlets in the middle.
Chemotherapy targets.
Tubulin: alpha tubulin and beta tubulin, dimer. Functionally building block of the microtubule.
Alpha and beta tubulin organized linearly to form one protofilament, 13 profilaments forms a microtubule. There’s a seam in the
middle.
Critical concentration: concentration above which the concentration would grow. There’s no fixed concentration for microtubule
because they are not as well known as actin.
GTP tubulin forms stable polymer. Likes to be in polymer, likes to be in tube. GDP doesn’t like to be in polymer, wants to get out,
it is kinked. In a catastrophe, the microtubule collapse.
Dynamic instability: GTP cap keeps the whole polymer stable.
-Microtubule-Associated Proteins
Microtubule organizing center: centrosome in animal cells. microtubule negative ends are embedded in it. Plus ends are in the
cytoplasm.
Centrosomes: mother centriole and daughter centriole.
Pericentriolar material: whatever things that surrounds the centrioles.
Microtubule nucleation factor: gama-TURC. It has red disks. Nucleation is very slow.
Microtubule-severing enzymes: catanin, it cuts microtubule.
Plus-TIP proteins: located at the plus end of the microtubule. EGFP-EB1 (end binding protein 1). Works as a marker to see the
plus ends of microtubule. CH domains: caponin-homology domains, binds only the plus ends of the microtubule. Recognizes the
GTPs. Other plus TIPs hitch a ride on EB1.
Depolymerases: kinesin 13 family proteins, which are part of the kinesin proteins. They break down physically the ladder by
using energy of ATP. Stathmin grabs four tubulin in solution and not letting them get out, keeping the tubulin not able to stick
back on to the microtubule. MCAK: a kind of kinesin 13 protein. The control filament has a blunt end. But, when +MCAK is added,
the end becomes open.
Polymerases: microtubule polymerase act like RNA polymerase. as concentration of tubulin increases, growth rate increases.
Growth rate as a function of concentration. XMAP215 increases K on of tubulin by factor of 5. XMAP 215 binds to tubulin. All of
the polymerases act by the same principle. Binds to monomer, and stick them together. Add tubulin to the end of microtubule.
RNA polymerase, DNA polymerase, formin act the same way. It’s like a catalyst. Catalyze both the forward reaction and reverse
reaction. Catalyzes the reverse reaction. Chimography. Putting a movie into an image. The length of the microtubule decreases
when there’s 0 concentration of tubulin (no free tubulin). When a little of tubulin is added, stoped depolarizing. XMAP 215 also
stabilizes the tubulin when it is first added on to the microtubule.
Lecture 26
Long range transport is CONTROLLED. A squid has a big axon.
Kinesin is a dimer, 45 kinds. They “walk” on beta tubulin of microtubule towards the plus end, compared to myosin, walks on
actin filaments.
0, forward:ADP; backward: ADP
1, forward: empty, binds to beta tubulin. Backward: ADP
2, conformational change: forward: ATP, swings backward to become the new forward (ADP).
3, forward (ADP) releases, backward(ATP) hydrolyzes to ADP to Pi.
Dyneins are minus end directed, they have dynactin binding domain, ATPase domain and microtubule binding domain, unlike
kinesin, the microtubule biding domain and ATPase domain are together on the head of the kinesin.
There’re adapter proteins that link cargos to different proteins.
Flagella has nine plus two arrangement. Nine doublets plus two singlets. The doublets are linked by nexin.
Lecture 27:
Cancer cells can divide without stop. They do not require any substrate to divide.
Half life is 4 minutes. Half life in microtubule spindles during mitosis is about 15 seconds.
XMAP215: zenopeuse microtubule associated protein with 215 molecular weight.
Mitotic chromosome has two chromatids stuck together. They are stuck together at kinetochore.
Keep the replicated chromosomes together.
Forces are pulling on kinetochore on each side. They will reach balance at one point and at this point, the metaphase plate is
formed. All chromosomes match up at equator. All of the microtubules are tread-milling.
One thing to stop cancer is to disrupt mitosis.
Growth and shrinkage of microtubules: dynamic.
Kinesin 13 depolamerizing kinesin. It mediated depolymerization of microtubule at their plus ends. The dynein mediated minus
end directed walking, dynein pulls the microtubule towards all the minus ends. On the chromosome: chromo kinesin which is
Kinesin 4 is plus end directed motor. It’s pulling the chromosome towards the metaphase plate in the center where all the plus
ends are. That’s why these chromosomes are always pointed towards equator even when anaphase happens. Pole ejection force,
contradicted to dynein, it’s pulling away from the poles. Kinesin 4 gets stuck on the plus end of the microtubule as the
microtubule grows, so it keeps being stuck on the plus end.
Lecture 28:
Cortex: area under the plasma membrane that has a high concentration of actins microfilaments in it.
Astral microtubules will be in the cortex. The minus ends are in the centrosomes, plus ends are in the spindle. The polar fibers
are anti-polar. The kinetochore gets stuck to the microtubule in such a way that the microtubule
Four proteins during anaphase:
1. Kinesin 13: depolarize
2. Kinesin 7: hangs on as the microtubule grows
3. Kinesin 4 pulls the chromosome towards the equator where all the plus ends are.
4. Dynesin pulls the chromosome towards the minus ends where the centrosome are.
Mitosis is part of Cell cycle, it refers to the passage of cells in their lifes through mitosis into their life as a new cell, their
replication of DNA and then mitosis again. Most cells that are proliferating are called stem cells. Lots of stem cells are
constantly going through cell cycle. Bone marrow, skin, gut lining, brain. Cells are nerve cells that are like nuclei. Cranial
radiation is the result of cell cycle deficiency.
Mitosis: cell undergoes mitosis forming two new cells.
Interphase is the time after cytokinesis until the next time it enters mitosis.
Interphase consists of three different segments. Immediately after division, the cell is in G1, gap 1. Cells do not start
duplicating themselves immediately after cytokinesis. They grow, get bigger and bigger, in fact, the time that they’re gonna
divide is determined by the size they reach. Once they reach a certain size, they commit to division, and they go through rest
of the cycle. If we start the cells, and they can’t reach that size, they cannot go through division and they don’t go through
mitosis.
The cell cycle consists of four phases: M, G1, S, G2 and back into mitosis. The cell cycle like this needs tight regulation.
Restriction point: at this point, the cell continues for sure. The chromosome would start replicating. When it enters S phase,
it starts to replicate its chromosomes. Needs tight regulation and all different stages of regulation.
Spindle assembly check point: checks if the cell is alright. If not alright, goes back for repair. Mitosis’s about an hour. G0 are
for cells that are damaged.
Three ways that the cell cycle is regulated:
1. Heterodimeric kinases
Two different subunits. Kinase subunit and a regulatory subunit. The kinase has to bind to the regulatory unit. Rather the
regulatory subunit is available or it is activated. It acts as a control.
2. Protein degradation: it’s one way direction. Can not go the reverse way. Can not be reversible. Two or three points in
the cycle, two or three proteins are broken down. They’re not there anymore so they can not influence the cycle so the
cycle has to go forward.
3. Checkpoints: points in the cycle where we surveying the cycle, if things are going wrong, we stop it. If it can’t be fixed,
we kill the cell, if we can fix it, it goes on. The three main drivers for checkpoints: (I) DNA damage is untolerable, (II)
unreplicated DNA, (III) unattached kinetochores (not evenly distribute chromosomes).
Amphibian egg maturation: all of the original works are done in the first four species. It is a highly conserved system. We
still retain the same machinery. Froggy eggs are big. So we can get a lot of stuff out of them.
The cell goes on mitosis and S phase for like 12 times, then when we put in progesterone, it goes through meiosis I, but
arrested in G2 first.
MPF: mitosis promoting factor, inject it into a cell and drive it into meiosis. Directly affect the cell if it goes into mitosis
or meiosis. We don’t know its molecular identity. It is an activity.
Cyclin: cyclically going up and down. Shortly after cyclin peaks, cells start to divide, undergoes division. As cyclin
decreases, cells leave mitosis, leaves division. The increase in cyclin is correlated with cells entering mitosis. The
decrease (breakdown) of cyclin (activity of the protein) is correlated with the cell leaving mitosis. Sea urchin cyclin is
similar to frog cyclin. The antibody of sea urchin cyclin binds to frog MPF (frog cyclin). This is called conservation of
proteins in the cell cycle.
Cyclin: When the cell enters mitosis, cyclin builds up, when the cell leaves mitosis, cyclin disappears. Frog MPF is
recognized by sea urchin antibodies.
If treated with RNase, cyclin B never turns up, no MPF, no mitotic events happening. Without the synthesis at least of
cyclin B, no entry into mitosis. If take this preparation, but add back cyclin B, the complete process is reconstituting. It’s
the single component that’s necessary for mitosis.
Lecture 29:
In order to go into mitosis, we need cyclin B, in order to get out of mitosis; we need cyclin B to be broken down. G2:
checkpoints, if the cell is damaged, the cell is not going through mitosis.
During G1, daughter cell grows. A bud emerges when the cell hits S phase, then the cell grows gradually until G2.
Cell cycle mutant: cell cycle mutant at the particular point of a cycle. Some yeasts are dividing, some mutations called
wee mutations. The cells come out very very small.
Cdc: cell division cycle. The cell keeps growing but never gets divided. There’s neither mitosis nor nuclear division
occurring. We can prevent the cells from entering mitosis by mutating this kinase. The kinase is homologous would bind
and recognize MPF from a frog. This kinase is one of the molecules in the MPF.
MPF: an activity that drives cells into mitosis and holds them there. Heterodimer: cyclin, and a kinase, (cyclin and cyclin
dependent kinase).
Cdc2 molecule is also homologous in the human being. MPF is conserved from yeast to human.
Cdk: cyclin dependent kinase. T loop. When it binds to T loop, stuffed into its active site. The active site is blocked by the
T loop. When it binds to cyclin, the T loop flips out and exposes the active site, somewhat activating the cyclin
dependent kinase. But not completely activated at this point. In order to be completely activated, a site on the T loop
has to be phosphorylated by CAK (cyclin depending kinase activating kinase). That’s active MPF.
Anywhere we look in living system, we always find activation balanced by inhibition. Can not have one without the other.
Other phosphorylation sites on the kinase. Wee1 kinase (inactivates MPF) by phosphorylating the cdk1 and CAK kinase
(activates the MPF). If we knock out wee1 kinase, we can no longer inhibit MPF. Once we knock out the kinase, the MPF
is free willing, not regulated anymore, so the cell proliferates more and more frequently. This is the target of wee1
mutation. It’s an inhibitory kinase. It shuts down MPF. If the MPF is too active, the cell keeps undergoing mitosis.
The other way to block or regulate the activity of the kinase (CDK) is by binding inhibitory molecules to it. Two different
inhibitory molecules: kinase or the complex( the dimer: kinase and cyclin), both of them either blocks the active site or
interfere its function. Either phosphorylate them or bind inhibitor to them. In both case, inhibition occurs.
Only need MPF to function at one small point during the cycle. During G1 or S, we break it down.
Inhibition: (I)Inhibitory phosphorylations (II) Bind inhibitor (III) Break down components (break down cyclin B in case of
MPF).
Activation: (I) binding cyclin (II) activating phosphorylation
It’s a heterodimeric kinase. As soon as cyclin B starts rising, the complexes with the kinase with the Cdk, immediately
Cdc25: This phosphatase specifically removes the inhibitory phosphates.
Get out of mitosis: APC anaphase promoting complex. Gets the cell through anaphase. One of one of the major ways is
to break down cyclin. APC takes ubiquitain (marker for proteins that are gonna be degraded) and sticks it on to
particular proteins, targeting them for degradation. The reason cyclin B gets broken down is because APC ubiquitin
makes it. It sticks a chain of ubiquitins on to cyclin B, and cyclin B is then broken down by the cell machinery. APC is not
random, first of all, it’s activated by MPF which phosphorylates and activates it. MPF activates the mechanism that shuts
itself off. APC has to be targeted. The early targets and the late targets: early targets are mediated through a molecule
called cdc20, cdc20 binds to APC, targets it to the molecules holding the chromosomes together so they can be broken
down so the chromosomes can be separate.
Cdc 20 is necessary for anaphase to start. It targets the anaphase promoting complex to the molecules holding the
chromosomes (sister chromatids) together. APC ubiquinates those proteins, those molecules, they get broken down, the
chromosomes are no longer held together so they pull apart. That’s how anaphase starts, through protein broken down.
Cdc 20 targets the APC is inhibited as long as there’s even one kinetochore unoccupied by microtubules. As long as
there’s one naked kinetochore, cdc20 can’t be activated. Spindle assembly checkpoint: system reading state of the
spindle. As long as even one chromosome that doesn’t have kinetochore occupied by microtubules, mitosis can not
continue. The reason why it can’t continue, cdc20 can’t bind to APC. An unoccupied kinetochore blocks the function of
cdc20.
APC: anaphase promoting complex.
Ubiquitin: marker for proteins that are gonna be degraded.
Cyclin B: the target protein to be degraded.
Maintains cycle in one direction (unidirectional).
Lecture 30:
Ubiquitin, any cell that has ubiquitin on it would be degraded. It’s a marker.
A ubiquitin ligase: an enzyme that links the ubiquitin to the target protein destruction box. It has recognition site on a
protein for ubiquitin ligase.
APC: a ubiquitin ligase that is called anaphase promoting complex. \
Cdc20: an accessory molecule that targets APC to securin.
Cdh1: an accessory molecule that targets APC to mitotic cyclin (B-type cyclins) and cdc20. When it’s phosphorylated, it’s
inactive, it’s phosphorylated for most of the cell cycle. Can be dephosphorylated by cdc14, it activates APC to break
down mitotic cyclins. Then drive the cell out of mitosis.
The only thing that allows anaphase to occur is separase: it can break down cohesin, allow the chromosomes to get
pulled apart finally. Normally, Securin is blocking separase. But when APC is targeted by cdc20, it breaks up, digests,
hydrolyzes securin, now separase is activated, anaphase can begin.
Comparing cell cycle of yeast and mammalian cell: cyclin D-CDK combination.
There’re lots of heterodimers here.
In yeast, only one cdk: cdc2, cyclin depending kinase. All of the unique targets of that kinase are determined by the
several different cdks that it can bind to. Several different cyclin depending kinases and those cyclines determine where
the single kinase directs its phosphorylations.
In mammals, there’re several different cyclins, also several different kinases as well. All of the cyclins have destruction
boxes. All three are broken down when the cell hits anaphase, and cdh1 binds to APC. Right after mitosis, there’s no
kinase activity at all. It’s a kinase free environment. Cdc14 which is a phosphatase, it’s anti-phosphorylation environment.
During that period, two things happen, first of all, APC remains active and targeted on mitotic cyclins. So mitotic cyclins
can build up during that period. Second of all, pre-replication complexes that licence DNA origins of replications to start
DNA replication. So during kinase free period, the cell is licencing its DNA replication origin, so when the cell hits S phase,
it can start replicating DNA. So the first step of S phase is actually taken up here where there’s no kinase activity around.
In the mammals, during G1 the cell could escape into G0 and drop out of the cycle.
Mid and late cyclin CDK dimers. Both are involved in setting the cell up and getting into S phase.
The cell is mediated by mid G1 CDK cyclin, then activate late G1 CDK cyclin, and then activate other components, the S
phase CDK cyclins and the DNA synthesizing enzymes. The important consideration with regard to the yeast situation is
that transcription of the mid G1 cyclin is determined by the nutrient level of the cell. It has a feed back mechanism that
responds to the level of nutrients in the environment. If it is well fed, it’s growing, then it starts producing the mid G1
cyclin CDK dimer, and the cell moves through the cell cycle. If we starve it, we can’t produce cyclin, then we can’t get
past the start point here, and it can’t enter the rest of the cell cycle. The reason the cell needs to grow to a certain size
before they divide is because the level of nutrients is what setting the expression of these first activators of the cell cycle:
the mid G1 cyclin CDK dimers. The final point to make is, we’re now activating these kinases, and CDH1 which is
phosphorylated, now inactive, can’t bind to APC anymore.
SPF: S-phase promoting factor, formally very similar to MPF. if it’s yeast, it would have the same cdk. Now we have the
G1 actin that’s active, the G1 cyclin cdk dimers, that drive the expression of S phase cyclin that immediately complexes
with the cdk. Now the cell should go into S phase, but it also immediately complexes with an inhibitor, sic1. As long as
it’s bound like that, the kinase activity is inhibited. Cdk kinase activity inhibitor: sic1. Similar to mitosis: wee1
phosphorylation that inactivates the dimer, the CAK phosphorylation that activates then we just build up a stock pile
finally just before we need it, we use phosphoatase to get rid of wee1 phosphate now we have a whole bunch of
activated MPF. S phase cyclin cdk is a bit slower, it’s held inactive by sic1, sic1 is a very weak kinase target for the G1
cyclins. So very slowly over G1, sic1 gets phosphorylated. Not until the very end of G1, sic1 is sufficiently phosphorylated
to become a target for polyubiquination, for ubiquin ligase.
What does S phase entail? It involves cell replicating its DNA.G2 M: As soon as the cell replicates its DNA, all of the
origins of replication (in bacteria, prokaryotes, there’s one origin of replication, in vertebrates and other animals with
large genomes, we have 46 chromosomes, therefore thousands of origin of replication on our DNA). So it goes much
faster. So those origins are always occupied by origin recognition complex. Basicaly the origins are occupied in that
fashion throughout the cell cycle. As the cell comes through mitosis, a whole series of proteins assemble on those
origins. The DNA has just been replicated, the instant the origins are replicated, the new origin activating complex
condenses or accumulates on it. In the kinase free environment in early G1, these kinase free subunits assemble on
origin recognition complex. It assembles the pre replication complex. So the Pre replication complex consists of the
origin replication complex and a bunch of proteins that are involved in DNA synthesis. But, some proteins actively
prevent DNA synthesis.the usual trick: we’ve accumulated the pre replication complex, the origin is ready to start to
replicate DNA, but it’s being held back. These two protein complex are hexamers are the helocase that wil unwind DNA.
They’re inactive, and there’re other proteins here as well. As the cell enters S phase, suddenly phosphorylation is the
name of the game. The S phase cdk dimers target these pre replication complex proteins and aggregates. And when they
do that, two things happen: some become activated like helocases, some inhibitory ones are released as is the
phosphorylated origin recognition complex. So the result of the phosphorylation by S phase dimers, replication can now
begin. The pre replication compelxes are built over the kinase free environment of early G1, then as phosphorylations
begin during S phase with the SPF, during that period, the components are phosphorylated and replication is activated.
We set things up as we might need with MPF and SPF, now with Origin of replication. In each case we do that, and when
we want to start the system, we have everything waiting, we phosphorylate, and then the system starts immediately
and runs full tail instead of building up slowly. we don’t want the cells dividing if it’s not big enough. This situation is
retained until the S phase cyclin is produced and activated at the beginning of S phase, and only then are these release
allow replication to occur. As long as everything is phosphorylated here, the system is replicating DNA. The origin
replication complex is immediately reformed on the origins. But DNA synthesis continues. As long as these components
are dissociated, are still phosphorylated, they can not reassemble. We can not reassemble pre replication complexes
while kinases are active. The only time the pre replication complexes can assemble is in the kinase free period of early
G1. Which means, we can only replicate DNA one time per cycle. Preventing mix up from occuring. The fundamental
reason why DNA replication happens only once in a cell cycle. As soon as the cell gets through mitosis, not even enter
the kinase free early G1, it’s a period with a lot of phosphotase activity. Cdc14 is active then. All the components are
dephosphorylated, and they accumulate or assemble at the origin recognition sites as the pre replication complex.
Overview of cell cycle: late G1 cyclins and DNA
Lecture 31:
Cell cycle and checkpoints:
The restriction point in mammals and start in yeast are the points in common. Once they pass this point, they’re
committed to one point.
B-type cyclins: the cyclins which transcription is driven by G1 cyclins: mid and late G1 cyclisn drive the expression the S
phase and M phase cyclins (B type cyclins). They contain the destruction box, so they’re targets for ubiquitin ligase, so
APC can break both of them down. When we get to the end of mitosis, we end up in the kinase free cyclin free period at
the beginning of G1. When S phase cyclin is first produced, it complexes with CDK, so it forms SPF, S phase promoting
factor. However, it’s immediately bound with sic1, cyclin depending kinase inhibitor, so it’s inactive, it only becomes
active late in G1 when sic1 becomes heaveily phosphorilated, and therefore the target of another ubiquitin ligase and it
gets broken down releasing a large burst of S phase promoting factor.
Hormones that get diffused throughout the body, affect various cells through various receptors. There’re three basic
basic type of compounds that function as hormones. Lipid soluble hormones, include steroids and other hydrophobic
molecules, steroid hormones, vitamin A, vitamin D, steroids, thyroid hormones, unique signalling mechanism that they
use. The other type is water, they’re polar. This includes modified amino acide like epinephrine or adrenaline, also,
there’re a lot of peptides, like insulin, glucagon, good way to deal with cells than the lipid hormone.
There’re several different means by which signaling occurs in the body. The first is endocrine signaling, that’s how signal
get into the blood, and an example is epinephrine. They have produced by adrenal gland. They have extraordinary
affects but very tiny amounts. The second type is paracrine signaling, that’s where the two cells that are having this
conversation are right next to each other. Here’s a cell that’s secreting hormone or signaling molecule, and here’s
another cell right next to it. These aqueous molecules cannot get through membrane. This is typical of nervous system.
Nerve growth factor, if added to the cell, they grow like nuts. Laminin is a protein, a structural protein. Extracellular
matrix can signal through these cells.
How to tell cells to do things? How to send signal from one cell to the other?
We do so through having ligands (signals) that bind to receptors. Those receptors produce internal signals. Those
internal signals transduce the extracellular hormone signal into the signal that means something for the cell. G protein
receptors are one kind. Other kinds of receptors: lipid soluble hormones (signaling molecules) can dissolve through
plasma membrane. So they do not have cell surface receptors. They dissolve through plasma membrane, their receptors
are transcription factors, and they bind to their receptor, move into the nucleus and activate transcription. Most of the
lipid soluble ligands that diffuse through plasma membrane activate transcription. First of all, their response is slow to
develop, (takes a while for transcription and translation to take place and produce an effect); second of all, they produce
persistent affect (if protein is produced, they will stay around for a while); and lastly, we don’t have extracellular affects
to break down ligand. They produce long lasting slow effects. They do so through activating transcription. The aqueous,
the hydrophilic ligands do not get across the membrane, they bind to their receptors. Three types of receptors.
1. G protein coupled receptor, bind to ligand.
2. Receptor protein kinase
3. Ion channel
G protein coupled receptor binds to ligand. Their receptor has seven trans-membrane segments. The G protein is this three
subunit, green protein down here. This is the protein that carries the signal inside the cell. On the other hand, the receptor
protein kinases bind ligand and their cytoplasmic end has kinase activity but it’s only activated when the receptor binds ligand.
They’re ligand activated enzymes. They’re not just kinases, they’re tyrosin kinases. The only amino acide that they phosphorylate
is tyrosin. The lipid soluble hormones: steroids, Vitamin D, Vitamin A, all the steroid hormones come from cholesterol. The three
major hormones: estrogen, testosterone, cortison, these hormones have major affects. Also, vitamin D is also produced by
cholesterol by acting on sunlight in the skin. Vitamin D is involved in bone mechanism, being taken seriously. It’s always being
thougth that it has something to do with viruses. Estrogen and testestorne involved in producing secondary sex characteristics.
Cotison is a stress hormone that’s involved in raising blood pressure and blood sugar. It’s also amino suppressive. these
hormones are present in the body in miniscule amounts.
Athletic ability: brings androgen back from Russia to United States. A lot of side affects. Severe heart attacks, paradoxically,
negative feedback. Too much testerosterone also make the body to produce estrogen. Too much estradiol would also make the
body to produce male sex characteristics.
Lipid soluble hormones:
We have receptors for the all of the steroid hormones. These receptors are inside the cell, the hormone diffuse is through the
cell. Retinoic acid is vitamin A derivative that’s involved in embryonic development. It’s also used for trying to cure athene. The
receptor for all of these receptors has similar structure: carboxyl binding domain, there’s ligand binding domain (50-70%
identical), at the other end at the center there’s DNA binding domain (90% homologous between them, similar to one another).
The amino terminal is variable, very different, it’s an activation domain, it’s related to binding, (Transcription factor, it binds
other cofactors to start transcription.) so these variable activation are involved in binding the particular co factors to are gonna
mediate the specific response to each of these signals.
One class is cytoplasmic, it’s inhibited. They work as mirror image dimmers with each other. Ligand binding domain and
activation domain. They’re homodimer. The response element will have DNA recognition sequence that’s facing the opposite as
well.
When estrogen is added, everything moves down into the nucleus. The cytoplasmic receptor binds to ligand, moves into the
nucleus, binds to appropriate response elements in the DNA, triggers transcription associated with the particular ligand that
used and the particular receptor. So we can look at the Zinc Finger Motif. The zinc finger transcription factor. The alpha helix is
binding to appropriate sequence in the DNA. If we look at the sequences, the response elements of the sequences, the different
types of hormone receptors for steroids and for lipid soluble homonres fall naturaly into different classes. The estrogen receptor
elements.
If we take a look at the ligands, the recognition sequence is reversed. Cytoplasmic, they bind ligand, they dimerize, they move
into the nucleus and bind to their response elements. The other class of these receptors are the vitamin D receptors, thyroxine
and retinoic acid. They all have precisely the same recognition sequence as the estrogen response elements. They all have the
same recognition sequence, different ligand, different receptor. These are arranged end to end.
VDRE:
Type I of the hormone response elements: includes Glucoticoid response element and estrogen response element. They are
cytoplasmic, they bind to ligands. Dimerize, move into nucleus and bind to their response elements. Inverted repeats: 5’ to 3’ on
top, 5’ to 3’ in the bottom. GRE: glucoticoid response element; ERE: estrogen Response Element; they’re homodimers. when it’s
estrogen receptor, it’s estrogen receptor estrogen receptor homodimer.
Type II: Vitamin D, thyroxin and retinoic acid. They have the same recognition sequence as the estrogen response elements.
Direct repeats: 5’ to 3’ on top and again 5’ to 3’ on top. they form RXR: All of these types form heterodimers like RXR: RX
receptors. Single molecule, a single receptor, each of them form a heterodimer. Vitamin D receptor, when it binds in the nucleus,
it’s vitamin D receptor : RXR, a heterodimer. It’s all heterodimer using RXR as the second component. Instead of head to head,
it’s head to tail. it’s the same orientation instead of different orientation. There’s one general RXR only. These receptors complex
are in the nucleus, they’re on the promoter of the gene they control, block transcription, so they’re there all the time, no
diffusion into the nucleus, they’re transcriptional inhibitors. When ligand gets in there, it diffuses into the nucleus, binds to
vitamin D receptor, then become an activator.
G protein coupled receptors:
G protein coupled receptors and cyclic AMP. They’re cell surface receptors. There’re 900 in the genome. They’re all over the
place. They’re involved in intercellular signaling, vision, olfaction, smelling, plays a major role in our life. 50% of the drug are
directed in the G proteins. As a result of conformational changes, rapid metabolism functions. The hemotaxis is related to G
protein coupled receptors. It’s really fast response. Also, longer term response mediated as with steroids by transcriptional
effects.
It has seven subunits. A cytoplasmic part and a nuclear part. These receptors interact with G protein. They’re active when they
bind GTP, they’re inactivate when they bind GDP. They’re trimeric subunit: alpha, beta and gama. The alpha subunit is the one
that carries the nucleotide: GTP or GDP, it gets activated and inactivated. The second and third subunit beta and gama get stuck
together permanently, act as one subunit.
Lecture 32:
The steroid hormone receptors are transcriptional factors that live in the cytoplasm. Under normal condition, they have their
inhibitor binding at the ligand binding domain, but when a ligand comes in and binds to the ligand binding domain, the receptor
gets activated by rejecting the inhibitor and gets into the nucleus and then gets dimerized and its DNA-binding domain binds to
the response elements in the DNA and therefore start transcription of the DNA. All of these receptors dimerize when they bind
to ligand, there’re two types of them.
G protein coupled receptors are for proteins that cannot get through the lipid membrane, the hydrophobic ones.:
GPCR: In G protein coupled receptor system:
three basic components:
1. the inactive receptor (epinephrein receptor, integral membrane protein that extends on extracellular and cytoplasmic
side of membrane)
2. the trimeric G protein with 3 subunits, stuck in the inner membrane by two lipid tail covalently bond. G alpha : GPTase,
bind GDP or GTP, it’s activated when bind to GTP, inactive to GDP.
3. the effector (an enzyme or an ion channel)
we want to link them together.
a. GEF: guanine nucleotide exchange factor
b. GAP: GTPase Activating Protein
c. RAS: small protein, bind GTP exchange for GDP, but not the GTPase activity.
Lecture of March. 29
G-ptorein adaptation: When we overstimulate a G protein, things happen to regulate its activity: always being controlled.
1. Always being controlled. Becomes phosphorylated by a dedicated kinase that associated with that receptor. Once
the receptor binds to the ligand, it interacts with its kinase and becomes phosphorylated. GRK: G protein coupled
protein kinase. The one we talked about is beta adrenergic receptor kinase. Each receptor has its own kinase, it gets
phosphorylated, binds arrestin, which interferes with its binding of ligand, also prevents from binding with the G
protein. Right away it’s being desensitized. It’s desensitized by its own kinase: homologous desensitization. Arrestin
is also the target for clathrin, they get endocytosed, either get recycled to the surface or broken down. The longer
we over stimulate, the more endocytoses we have, the less receptor we have, the less sensitive is the cell. So when
we flat the cell with ligand, it turns down its receptors. If we continue to flat it, we also get heterologous
desensitization. It’s just PKA phosphorylates all the GPCRs in that cell.
a. Homologous desensitization: associated with endocytosis, receptor specific.
b. Heterologous desensitization: not associated with endocytosis, universal G protein receptors.
In any events, the more we stimulate ,the more we turn things down
The other thing is: we’re hooked up to G alpha proteins that activate Phospholipase C, it cleaves phosphotidyl inositol
bisphosphate (PIP2 ), release IP3 (inositol trisphosphate) and diacylglycerol, the two of which can bind to PKC and drive
response. The initial calcium comes from ER, formally, it’s similar to muscle cells where action potential from nerve fiber invades
cytoplasmic reticulum and
Phospholipase C: cleaves PIP2
PIP2: releases IP3 (inside and DAG(membrane)
NOPKC: bind to IP3 and DAG, drive response.
Tyrosine kinase receptors: enzymatic receptors, when activated, have enzyme activity, specifically they’re kinases that transfer
gamma phosphate from ATP to tyrosines. The other kinases have been serine and threonine kinases. When get disrupted, we
get cancer.
They dimerize when they bind to the ligand.
Cancer:
Cancer cells don’t need promoting. Oncogenes that drive the cell cycle and are positively regulated. They’re genes that are
overdriven. Cancer cells are immortal.
Hela cells:
Self surface adhesion molecules
Nothing can deal with the spread of the cancer cells.