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We're going to continue our
discussion today about cell biology,

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and specifically about the subject
of signalling which we talked about

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in general terms last time.
I gave you some of the general

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principles about signaling,
signal propagation and signal

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processing in biological systems.
And today we're going to talk about

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two specific examples.
First a short-range response which

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results in the production of second
messenger cyclic AMP and the results

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of that in terms of downstream
events.  And the second will be an

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example of a long-range response
which requires new gene expression,

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specifically in the stimulation of
cells to divide.

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And the example I'll give you in
the context of wound healing.

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So just to finish up what I started
at the end of last lecture.

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As I mentioned,

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the energy associated with the
binding of a single ligand to a

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single receptor molecule at the
cell's surface is insufficient to

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trigger any of the downstream events
that are necessary either for the

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short-term or long-term responses.
And so it's necessary that the

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signal be amplified.
And there are various ways that

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this happens.  The first,
which I mentioned last time,

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is the production of molecules which
we refer to as second messengers.

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Second because they are not the
primary thing.

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The primary thing is the ligand
binding to the receptor.

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They come after that messenger
because they transmit the message

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related to the binding of the ligand
to the receptor inside the cell.

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And the example that I mentioned
and that we'll talk about in more

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detail today is a second messenger
called cyclic AMP or cAMP.

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I also mentioned that there are
examples of enzymatic cascades.

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And, again, the purpose here is to
amplify the signal.

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One enzyme turns on another enzyme
which turns on many more enzymes,

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et cetera.  It's like a pyramid
effect where something little grows

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to something big.
And the example I gave you last

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time was the analogy to dominos.
Pushing one over basically spreads

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the signal down the chain.
So signal amplification with

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respect to second messengers,
the principle is quite simple.

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Imagine an enzyme which is in an
inactive state.

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The binding of ligand to receptor
makes that enzyme go

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to an active state.
And in its active state it can

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convert a substrate into a product.
Now, if that product can stimulate

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a second enzyme,
imagine enzyme two,

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which is in an inactive state now in
the presence of this product,

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is converted to an active state,
you've amplified the signal.

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You've turned on a lot of enzymes
which make even more of this product

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which now stimulate yet another
enzyme.  So this process then

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results in a form of signal
amplification.

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The same is true for enzyme
cascades.  If you imagine an enzyme

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which is in an inactive state.
The binding of a ligand to a

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receptor converts that enzyme
to an active state.

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If that enzyme now acts on another
enzyme, E2 goes from inactive state

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to an active state,
and that enzyme then acts on another

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enzyme, E3 goes from an inactive
state to an active state,

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again, you've gone from a relatively
small amount of stimulus propagated

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through the stimulation of this
enzymatic activity,

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which acts on a number of these to
make even more of these active,

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which acts on even more of these to
make even more of these active,

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again, one develops signal
amplification.  OK?

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Now, these changes that take place,

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both in the generation of second
messenger, as well as in the turning

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on of enzymes,
often result from changes in protein

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states.  I tried to give you
indications last time that proteins

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are not static things.
They can move, they can interact,

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and they can actually change shape.
And changes in protein states are

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often reflected in changes in
protein structure,

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subtle changes in protein structure,
but nevertheless changes in protein

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structure.
And these can come in noncovalent

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forms.  The protein is not changed
by any new covalent bonds but by

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binding to something in a
noncovalent interaction.

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And the classic example of this are
G-binding proteins.

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These proteins bind guanine

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nucleotides.  That's why they're
called G-binding proteins.

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And they're often shortened and
referred to as G proteins.

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We'll meet two G proteins later in
today's lecture.

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G proteins change their structure
depending on whether or not they're

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bound to GTP or GDP.
Typically, in their inactive state,

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they're bound to GDP.  However, in
response to a signal transduction

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pathway, there's an exchange event
whereby GTP binds to the molecule

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and GDP is released.
This reaction is usually catalyzed

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by another protein known as a
nucleotide exchange factor.

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We're exchanging this nucleotide
for this nucleotide,

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and in so doing we change the shape
of the molecule.

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When it's bound to GTP now it has a
different shape.

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And by virtue of this different
shape, it might now be able to

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interact preferentially with another
molecule.  So imagine that we have a

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signaling molecule which has a shape
that looks something like this.

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A signaling protein.

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Given the lack of complementarity
between this shape and this shape,

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these proteins will not interact.
However, upon nucleotide exchange

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such that the protein now binds GTP
and now changes its shape,

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now we can get a productive
interaction between the G protein

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and the signaling molecule.
And this can lead to a form of

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signal amplification.
OK?  So that's an example of a

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noncovalent change,
and a reversible one.

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The GTP can be hydrolyzed --

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-- back to GDP,
thereby shutting the signal off.

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OK?  Another example of a
structural change in a protein but

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now a covalent one --

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-- is in the form of phosphorylation.
So imagine that we have a protein

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here, an enzyme which is
in its inactive state.

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And this protein has among its amino
acids, in some critical position,

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an amino acid with a hydroxyl
residue.  Does anybody remember back

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in the Dark Ages when you were
learning your amino acid structures

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which amino acids have hydroxyl
residues?  I'm tempted to offer

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money to anybody who would remember
all three.  Anybody.

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Does anybody remember even one?
Serine it is.  Yes,

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that's one.  Serine.
Anything else?  Tyrosine is another.

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Very good.  And the last one?
Threonine.  So these three amino

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acids have hydroxyl residues.
And by virtue of that they are

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subject to, or can be subject to
protein phosphorylation at the hands

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of enzymes, which I've referred to
in previous lectures, called

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protein kinases.
These proteins can be modified such

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that they have a phosphate group on
them.  And this can change the

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structure of the protein and make it
go from an inactive to an active

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state.  There are actually examples
where it goes the other way,

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phosphorylation takes an active
protein and makes it inactive,

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but you see the point.
Phosphorylation can change the

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activity.  And,
once again, this is reversible.

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There are protein phosphatases
which will remove the phosphate and

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return the protein to its baseline
state.  OK?  So just to give you an

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example of how this
might be useful.

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Imagine that I have an enzyme here
which has an active site,

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but this active site, by virtue of
the confirmation of the protein,

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is closed.  That is the substrates
cannot get in there in order to be

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modified.  At the hands of a
specific kinase that might recognize

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a hydroxyl residue right here,
the shape of this protein might

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change.
So now the active site becomes open

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and the enzyme becomes active.
OK?  And likewise this can be

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reversed through the action of a
phosphatase.  OK?

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So protein structures change,
activities of proteins, enzymes

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change.  And that's one way in which
we can propagate signals.

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OK.  So let's now go to a specific
example.  And the example that I

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want to give you is one that
hopefully not many of you are

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familiar with.
If you're camping and you run

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across this fellow here,
what is your immediate reaction?

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You run.  You run. You never try to
feed the bear a marshmallow with

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your mouth.  That's the first thing
I learned about camping,

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you run.  This is the famous fight
or flight response.

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And when you're dealing with a bear,
you want to take the flight option.

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It's stimulated by fear,
some perception of fear.  And upon

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perception of fear --

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-- through a complex physiology,
a small molecule is released from

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your adrenal glands.
Glands that sit on top of your

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kidneys.  This molecule is called
epinephrine, otherwise known as

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adrenaline.  So this is released
from your adrenal glands and various

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things happen.
One, that we're going to spend a

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little time talking about,
is that your blood glucose levels go

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up.  Why?  Why do you want your
blood glucose levels to go up when

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you see this fellow here?
So you can run.

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Remember, that's the first thing you
want to do when you see a bear,

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run.  In addition, your heart rate
goes up so you can pump more blood

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to your big muscles,
your breathing rate goes up so you

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can oxygenate those muscles,
and your blood pressure goes up.

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And there are other things that
happen as well,

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but these are the most important
with respect to the fight

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and flight response.
This happens because this hormone,

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epinephrine, triggers all of these
responses, actually in different

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cells within your body.
Because it's acting at distance

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sites within your body,
from the adrenal glands to the

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muscles to the liver to the heart to
your brain, this is referred to as

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an endocrine response.
An endocrine response is basically

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defined as hormones acting
at a distance.

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OK?  Produced somewhere.

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Acting somewhere else in your body.
We differentiate that with the

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paracrine response in which there is
a local release of growth factors

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and other ligands.

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So here there's a local issue.
Something happens locally, factors

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are released right in the
environment, and they act upon cells

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right in their neighborhood.
And finally a related turn called

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autocrine which is cells --

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-- which release the factor and bind
it themselves.

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Autocrine.  So they stimulate
themselves.  And very often you'll

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start a process one of these ways.
And then the cell will take over.

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Basically amplifying the signal for
itself.  OK?  Now,

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in the example that I want to talk
about in the first portion

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of the lecture.
We're going to deal with this one

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here, blood glucose,
the increase levels of blood glucose.

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As you probably know,

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your body stores energy different
ways.  One way is in a polymer of

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glucose called glycogen,
which is stored in your liver.

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Another way is through the storage
of fat.

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For this response we want to
liberate the energy that's stored in

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the form of glycogen and turn it
into glucose which is much more

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consumable. It can be used directly
by your muscles to make ATP which

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drives various reactions like
contractions and so on.

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So the analogy here is money in
your bank account versus

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money in your wallet.
When you're in a situation like that,

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you want to transfer money from your
bank account to your wallet.

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That's the direction we're heading
in this example.

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In others situations,
where you've just had a huge meal

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and you're not running from a bear,
you go the other way.  You take

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glucose and you turn it into
glycogen, which I said is a polymer

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of glucose.  Now,
this balance between glycogen and

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glucose is controlled by enzymes
which are sort of in the center of

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today's example.
In this direction the enzyme is

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glycogen phosphorylase.
Glycogen phosphorylase is the last

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enzyme that breaks down glycogen
into a form of glucose that can be

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then further metabolized.
Glycogen phosphorylase.  And the

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other, in the other direction,
is an enzyme called glycogen

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synthase.  OK?
So the goal of this reaction,

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or set of signals, will be to
stimulate this process and actually

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inhibit this process.
And you'll see how that's done in a

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second.  This is epinephrine.
Again, it's a small chemical

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produced by your adrenal glands.
As I said earlier, it's released

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during the flight or fight response.
It binds to a class of receptors

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which I'll take you through.
They're called beta-adrenergic

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receptors
because they bind to a factor

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produced in the adrenal glands,
beta-adrenergic receptors.  These

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are receptors,
as you'll see, that have a complex

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tortuous root through the membrane.
They go through the membrane seven

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times.  They're actually sometimes
referred to as serpentine receptors

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for that reason.
And they bind to and activate GTP

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binding proteins,
G proteins, as I mentioned down here.

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And this is a particular form of G
protein called trimeric G proteins.

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And you'll see why in a second.
As I mentioned,

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epinephrine binds to cells in
different parts of the body

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resulting in various physiological
effects.  The ones I listed here and

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another one which I didn't list here
which I find paradoxical.

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Actually, I know what, I know what
the evolutionary advantage to all of

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these things is,
but I don't know what the

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evolutionary advantage is to the
relaxation of the smooth muscles

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around the colon during the fight or
flight response.

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Some of you may have felt this
before when you're extremely fearful.

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I'm not sure why it is
our bodies do that.

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But anyway.  OK.  So
how does this work?

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So we're going to consider just the

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mobilization of glucose.
And the site of action is the liver.

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00:19:52,000 --> 00:19:57,000
That's where glycogen is stored.
And on the cells in the liver, the

226
00:19:57,000 --> 00:20:02,000
hepatocytes, on the plasma membrane
of those cells,

227
00:20:02,000 --> 00:20:07,000
there are these receptors,
these serpentine receptors.

228
00:20:07,000 --> 00:20:11,000
They look like that.
They look like snakes,

229
00:20:11,000 --> 00:20:16,000
as I mentioned.  Beta-adrenergic
receptors.  There are also

230
00:20:16,000 --> 00:20:20,000
alpha-adrenergic receptors,
but we're going to focus on

231
00:20:20,000 --> 00:20:25,000
beta-adrenergic receptors.
And these bind to epinephrine

232
00:20:25,000 --> 00:20:30,000
directly.  So here's our molecule
of epinephrine.

233
00:20:30,000 --> 00:20:37,000
It physically binds to a portion of

234
00:20:37,000 --> 00:20:43,000
the polypeptide chain that's
sticking out from the cell's surface.

235
00:20:43,000 --> 00:20:48,000
Now, bound on the inside to this
receptor is this trimeric G protein.

236
00:20:48,000 --> 00:20:54,000
It's called trimeric because there
are three subunits.

237
00:20:54,000 --> 00:21:00,000
There's an alpha subunit,
a beta subunit and a gamma subunit.

238
00:21:00,000 --> 00:21:06,000
And in this configuration,
prior to the binding of epinephrine,

239
00:21:06,000 --> 00:21:12,000
the alpha subunit is bound to GDP.
And, as I mentioned before,

240
00:21:12,000 --> 00:21:19,000
that's usually associated with
inactivity.  So the alpha subunit is

241
00:21:19,000 --> 00:21:25,000
inactive.  The binding of
epinephrine to the surface has two

242
00:21:25,000 --> 00:21:31,000
effects.  The first is an exchange
wherein the alpha subunit

243
00:21:31,000 --> 00:21:37,000
now binds GTP.
And the GDP that was present there

244
00:21:37,000 --> 00:21:42,000
comes off.  The other effect is that
the G protein,

245
00:21:42,000 --> 00:21:47,000
the alpha subunit releases from the
beta and gamma subunits and they

246
00:21:47,000 --> 00:21:53,000
float away.  In this,
now GTP-bound configuration,

247
00:21:53,000 --> 00:21:58,000
the alpha subunit is now active.
And it can move from this position

248
00:21:58,000 --> 00:22:03,000
to another position within the
membrane and bind to

249
00:22:03,000 --> 00:22:09,000
another protein.
In this case an enzyme called

250
00:22:09,000 --> 00:22:16,000
adenylate cyclase.
The GTP bound version of the alpha

251
00:22:16,000 --> 00:22:23,000
subunit associates with adenylate
cyclase, thereby converting it to an

252
00:22:23,000 --> 00:22:31,000
active form.  When bound to this it
becomes active.

253
00:22:31,000 --> 00:22:37,000
And the consequence of that is that
the adenylate cyclase can now

254
00:22:37,000 --> 00:22:43,000
convert ATP to the second messenger
cyclic AMP.  OK?

255
00:22:43,000 --> 00:22:49,000
And this process that we've gone
through from binding of a single

256
00:22:49,000 --> 00:22:56,000
molecule to the production of a lot
of a cyclic AMP is an example.

257
00:22:56,000 --> 00:23:18,000
OK?  We've made a lot of second

258
00:23:18,000 --> 00:23:22,000
messenger from the binding of a
single molecule because we've turned

259
00:23:22,000 --> 00:23:25,000
on a lot of these,
which can go on and turn on a lot of

260
00:23:25,000 --> 00:23:29,000
these, which can go on and
make a lot of this.

261
00:23:29,000 --> 00:23:40,000
Now, there's another step in signal

262
00:23:40,000 --> 00:23:48,000
amplification here in this pathway.
There's an enzyme known as protein

263
00:23:48,000 --> 00:23:57,000
kinase A which becomes activated in
the presence of cyclic AMP.

264
00:23:57,000 --> 00:24:05,000
So protein kinase A in the presence
of cyclic AMP goes from an inactive

265
00:24:05,000 --> 00:24:13,000
state to an active state.
And when it becomes active it

266
00:24:13,000 --> 00:24:21,000
converts another enzyme,
glycogen phosphorylase, which I

267
00:24:21,000 --> 00:24:29,000
mentioned to you a moment ago,
from an inactive state to an active

268
00:24:29,000 --> 00:24:43,000
state.

269
00:24:43,000 --> 00:24:46,000
OK?  So this is a phosphorylation
reaction, similar to the one that I

270
00:24:46,000 --> 00:24:50,000
drew up here.  PKA phosphorylates
glycogen phosphorylase,

271
00:24:50,000 --> 00:24:54,000
making it go from an inactive to an
active state.  Another step of

272
00:24:54,000 --> 00:24:58,000
signal amplification.
We can make some of this active

273
00:24:58,000 --> 00:25:03,000
which makes a lot of this active.
OK?  And this is the enzyme we

274
00:25:03,000 --> 00:25:09,000
needed to turn on in order to break
down glycogen to glucose.

275
00:25:09,000 --> 00:25:16,000
Now, there's another kind of cool
thing that happens in the control of

276
00:25:16,000 --> 00:25:22,000
the reverse reaction,
because it turns out that the enzyme

277
00:25:22,000 --> 00:25:28,000
responsible for making glycogen,
glycogen synthase is inactivated in

278
00:25:28,000 --> 00:25:35,000
the presence of cyclic AMP.
So it goes from an active state in

279
00:25:35,000 --> 00:25:41,000
the presence of cyclic AMP
to an inactive state.

280
00:25:41,000 --> 00:25:49,000
So at once we turn that off,

281
00:25:49,000 --> 00:25:54,000
turn this on, and we can generate a
lot of glucose.

282
00:25:54,000 --> 00:25:59,000
OK.  So this is the reaction.
We focused exclusively on the

283
00:25:59,000 --> 00:26:03,000
mobilization of glucose here.
Just to remind you,

284
00:26:03,000 --> 00:26:06,000
this same pathway controls heart
rate, breathing rate,

285
00:26:06,000 --> 00:26:10,000
blood flow.  And, actually,
some of you might have heard of

286
00:26:10,000 --> 00:26:13,000
beta-blockers.
And beta-blockers are used to

287
00:26:13,000 --> 00:26:16,000
control this exact reaction.
Beta-blockers bind to

288
00:26:16,000 --> 00:26:19,000
beta-adrenergic receptors so that
your body won't respond to

289
00:26:19,000 --> 00:26:23,000
adrenaline or epinephrine when you
don't want it to.

290
00:26:23,000 --> 00:26:28,000
So people who get really nervous
talking in front of audiences or

291
00:26:28,000 --> 00:26:33,000
taking exams in 7.
1 will sometimes take beta-blockers

292
00:26:33,000 --> 00:26:38,000
so that they don't suffer,
you know, fast heart rate or this

293
00:26:38,000 --> 00:26:44,000
feeling of anxiety and so on.
OK.  So we've turned on this signal

294
00:26:44,000 --> 00:26:49,000
in response to binding of
epinephrine to the cell's surface.

295
00:26:49,000 --> 00:26:54,000
We've started all these happening.
How do we turn it off?  How do we

296
00:26:54,000 --> 00:27:00,000
deal with signal termination?
How do we turn the signal off?

297
00:27:00,000 --> 00:27:05,000
Anybody have any ideas?
Well, one might be not so obvious.

298
00:27:05,000 --> 00:27:16,000
And that is --

299
00:27:16,000 --> 00:27:19,000
-- a process which I introduced to
you last time called pathway

300
00:27:19,000 --> 00:27:23,000
modulation in which the activation
of a second pathway can actually

301
00:27:23,000 --> 00:27:27,000
inhibit the activation of
the primary pathway.

302
00:27:27,000 --> 00:27:32,000
So, for example,
I've been telling you about the

303
00:27:32,000 --> 00:27:37,000
beta-adrenergic receptors,
which through the binding of this

304
00:27:37,000 --> 00:27:42,000
kicks off a series of events which
results in the activation of this

305
00:27:42,000 --> 00:27:48,000
pathway.  There are many examples in
biology where the binding of a

306
00:27:48,000 --> 00:27:53,000
second ligand to a distinct receptor
results in the propagation of a

307
00:27:53,000 --> 00:27:59,000
signal which actually
inhibits the first.

308
00:27:59,000 --> 00:28:02,000
So, for example,
I mentioned here that there's a

309
00:28:02,000 --> 00:28:06,000
kinase that's activated,
protein kinase A.  This pathway

310
00:28:06,000 --> 00:28:10,000
might activate the relevant
phosphatase, thereby turning this

311
00:28:10,000 --> 00:28:13,000
signal down.  So modulation of the
pathway, very important in biology.

312
00:28:13,000 --> 00:28:17,000
We're only really beginning to
appreciate how important it is the

313
00:28:17,000 --> 00:28:21,000
more we learn.
Another obvious example is ligand

314
00:28:21,000 --> 00:28:25,000
diffusion.  You make a certain
amount of ligand from

315
00:28:25,000 --> 00:28:30,000
your adrenal glands.
And it just diffuses eventually or

316
00:28:30,000 --> 00:28:36,000
breaks down, thereby turning the
signal off.  There's also a

317
00:28:36,000 --> 00:28:43,000
phenomenon known as receptor
internalization.

318
00:28:43,000 --> 00:28:51,000
Very often when a receptor binds to

319
00:28:51,000 --> 00:28:55,000
its ligand, it doesn't just stay out
there on the surface but actually

320
00:28:55,000 --> 00:28:59,000
gets internalized inside the cell.
So I'll draw a different class of

321
00:28:59,000 --> 00:29:03,000
receptors here.
Here's the receptor.

322
00:29:03,000 --> 00:29:07,000
Here it's bound to its ligand.
I told you last time that proteins

323
00:29:07,000 --> 00:29:11,000
can get to the cell's surface
through a complex sorting pathway

324
00:29:11,000 --> 00:29:16,000
that involves actually vesicles
encapsulating the proteins and then

325
00:29:16,000 --> 00:29:20,000
bringing them to the cell surface.
Well, the reverse can happen as

326
00:29:20,000 --> 00:29:25,000
well where the proteins can be
removed from the cell's surface into

327
00:29:25,000 --> 00:29:29,000
vesicles.  Removing them from the
cell's surface then takes them away

328
00:29:29,000 --> 00:29:33,000
from the possibility of being
activated, and thereby turning

329
00:29:33,000 --> 00:29:38,000
down the signal.
Another possibility would be to

330
00:29:38,000 --> 00:29:43,000
degrade the second messenger.
OK?  Cyclic AMP is an example.

331
00:29:43,000 --> 00:29:47,000
You need to make it in order to
stimulate these various downstream

332
00:29:47,000 --> 00:29:52,000
events, so get rid of it.
And, in fact, there's a clear

333
00:29:52,000 --> 00:29:57,000
pathway for that process.
As I told you, ATP can be converted

334
00:29:57,000 --> 00:30:02,000
through the actions of adenylate
cyclase to make cyclic AMP.

335
00:30:02,000 --> 00:30:10,000
But likewise there's an enzyme,
actually, a whole class of enzymes

336
00:30:10,000 --> 00:30:18,000
known as phosphodiesterases which
converts cyclic AMP to AMP,

337
00:30:18,000 --> 00:30:26,000
thereby inactivating the signal.
These enzymes are actually very

338
00:30:26,000 --> 00:30:32,000
important in pharmacology.
There are lots of inhibitors of

339
00:30:32,000 --> 00:30:36,000
phosphodiesterases which stimulate
the production or the maintenance of

340
00:30:36,000 --> 00:30:40,000
high levels of cyclic AMP and other
cyclic nucleotides.

341
00:30:40,000 --> 00:30:44,000
So caffeine for example,
one of the reasons you get a buzz

342
00:30:44,000 --> 00:30:48,000
from caffeine is that it blocks a
class of these enzymes leading to

343
00:30:48,000 --> 00:30:52,000
more cyclic AMP which gives you that
kind of nervous energy.

344
00:30:52,000 --> 00:30:56,000
OK?  Another related process
involving another cyclic nucleotide,

345
00:30:56,000 --> 00:31:01,000
this time cyclic GTP.
This is broken down by a different

346
00:31:01,000 --> 00:31:07,000
phosphodiesterase.
In Viagra and like chemicals are

347
00:31:07,000 --> 00:31:14,000
inhibitors of this class of
phosphodiesterases,

348
00:31:14,000 --> 00:31:20,000
thereby leading to an over
stimulation of that pathway.

349
00:31:20,000 --> 00:31:27,000
And, finally, you can reverse the
structural changes that I mentioned

350
00:31:27,000 --> 00:31:33,000
previously.  So I said earlier that
enzymes can be activated

351
00:31:33,000 --> 00:31:42,000
through kinases.
And they can be inactivated by

352
00:31:42,000 --> 00:31:52,000
phosphatases.  I'm going to have to
move to a different board here.

353
00:31:52,000 --> 00:32:02,000
And G proteins which get activated
in the beginning of these processes,

354
00:32:02,000 --> 00:32:12,000
from a GDP bound form, through the
action of exchange factors to the

355
00:32:12,000 --> 00:32:22,000
GTP bound form,
can be inactivated by GTP hydrolysis.

356
00:32:22,000 --> 00:32:26,000
And this is often through proteins
called GAPs, which are called GTPase

357
00:32:26,000 --> 00:32:31,000
activating proteins.
They activate the GTP hydrolysis

358
00:32:31,000 --> 00:32:36,000
activity of the G protein,
thereby returning the G protein to

359
00:32:36,000 --> 00:32:40,000
its inactive state.
OK.  So that's the beta-adrenergic

360
00:32:40,000 --> 00:32:45,000
signaling pathway.
It's a classic.  You should know it

361
00:32:45,000 --> 00:32:50,000
in principle and in some detail.
It's covered extensively in your

362
00:32:50,000 --> 00:32:55,000
book.  Here's a figure
from your book.

363
00:32:55,000 --> 00:32:58,000
I won't go through the details of
this because we just did them on the

364
00:32:58,000 --> 00:33:01,000
board, but just so you know it's in
there.  Here's the production of the

365
00:33:01,000 --> 00:33:04,000
second messenger,
cyclic AMP.  And then it triggers

366
00:33:04,000 --> 00:33:07,000
this kinase cascade that I drew up
for you before,

367
00:33:07,000 --> 00:33:10,000
activation of PKA,
activation of glycogen phosphorylase,

368
00:33:10,000 --> 00:33:13,000
and the production ultimately of
glucose.  And here's the

369
00:33:13,000 --> 00:33:16,000
second messenger.
I'm pleased to see that I put this

370
00:33:16,000 --> 00:33:19,000
in the book, in my slide set last
year because the book had an

371
00:33:19,000 --> 00:33:23,000
incorrect structure for cyclic AMP
in the previous edition.

372
00:33:23,000 --> 00:33:26,000
I'm pleased to see they know have
corrected the structure.

373
00:33:26,000 --> 00:33:30,000
This is the actual proper structure
of cyclic AMP.

374
00:33:30,000 --> 00:33:34,000
OK.  So now want to change signaling
pathways and consider not what you

375
00:33:34,000 --> 00:33:39,000
do when you first encounter the bear,
but what do you do,

376
00:33:39,000 --> 00:33:44,000
or what does your body do if you
fail to successfully run from the

377
00:33:44,000 --> 00:33:49,000
bear?  So it's funny what you can
find on the Web,

378
00:33:49,000 --> 00:33:53,000
boy.  You can find anything you want
on the Web.  In fact,

379
00:33:53,000 --> 00:33:58,000
when I was looking up,
I was pretty sure that Viagra

380
00:33:58,000 --> 00:34:02,000
blocked phosphodiesterases.
I was pretty sure,

381
00:34:02,000 --> 00:34:06,000
but I wasn't certain so I did a Web
search.  This is true.

382
00:34:06,000 --> 00:34:10,000
I did a Web search.  And,
God, I cannot believe what I came

383
00:34:10,000 --> 00:34:14,000
across.  It's unbelievable.
But you can find some pretty nasty

384
00:34:14,000 --> 00:34:17,000
bear wounds on the Web as well,
and here's one of them.  So this guy

385
00:34:17,000 --> 00:34:21,000
didn't run fast enough.
So what's going to happen?

386
00:34:21,000 --> 00:34:25,000
What is his body going to do
following an injury such as that?

387
00:34:25,000 --> 00:34:29,000
Well, obviously he needs to heal
the wound.

388
00:34:29,000 --> 00:34:33,000
Your body is remarkable in its
ability to detect damage and fix it.

389
00:34:33,000 --> 00:34:37,000
So we're going to heal the wound.
And this is an example of one of

390
00:34:37,000 --> 00:34:42,000
these long-term effects of a
signaling pathway.

391
00:34:42,000 --> 00:34:46,000
Whereas, this was short-term,
everything was happening in the

392
00:34:46,000 --> 00:34:50,000
cytoplasm, generation of glucose to
be secreted.  This is a long-term

393
00:34:50,000 --> 00:34:55,000
effect.  The effect has to take
place ultimately in the nucleus

394
00:34:55,000 --> 00:34:59,000
because we need to turn on gene
expression to convince cells to

395
00:34:59,000 --> 00:35:04,000
divide and heal the wound.
This may be a troubling picture.

396
00:35:04,000 --> 00:35:10,000
Maybe I should go back to the bear.
There.  So if we imagine the skin,

397
00:35:10,000 --> 00:35:16,000
which is a tight complex of cells
forming a protective layer against

398
00:35:16,000 --> 00:35:21,000
the elements and so on keeping stuff
out that should be out,

399
00:35:21,000 --> 00:35:27,000
in that should be in.  In a wound
you breach that structure,

400
00:35:27,000 --> 00:35:33,000
you get a scratch.
And one of the things that happens,

401
00:35:33,000 --> 00:35:40,000
as you bleed into that wound, is
that small packets,

402
00:35:40,000 --> 00:35:48,000
former bits of cells called
platelets bind to the edges of that

403
00:35:48,000 --> 00:35:55,000
wound.  And the platelets then
release a factor imaginatively

404
00:35:55,000 --> 00:36:03,000
called platelet-derived
growth factor.

405
00:36:03,000 --> 00:36:09,000
And platelet-derived growth factor,
otherwise known as PDGF stimulates

406
00:36:09,000 --> 00:36:15,000
other cells to divide.
And specifically it stimulates

407
00:36:15,000 --> 00:36:22,000
cells called fibroblasts which sit
underneath these cells normally as

408
00:36:22,000 --> 00:36:28,000
part of the connective tissue
underneath the epithelial cells.

409
00:36:28,000 --> 00:36:35,000
In the presence of PDGF, the
fibroblasts proliferate.

410
00:36:35,000 --> 00:36:39,000
And this results in the formation of
a scar.  OK?  And what we want to

411
00:36:39,000 --> 00:36:44,000
understand now is how is it that
PDGF stimulates fibroblasts

412
00:36:44,000 --> 00:37:21,000
to divide?

413
00:37:21,000 --> 00:37:29,000
So we can model this process in cell
culture.  We can extract fibroblasts

414
00:37:29,000 --> 00:37:37,000
from your skin and put them in a
tissue culture dish,

415
00:37:37,000 --> 00:37:45,000
let's say one times ten to the fifth
cells.

416
00:37:45,000 --> 00:37:51,000
So this is cell number on this axis
and this is days in culture on this

417
00:37:51,000 --> 00:37:57,000
axis, one, two,
three, four days.

418
00:37:57,000 --> 00:38:03,000
If we plate 100,000 fibroblasts in
a tissue culture dish,

419
00:38:03,000 --> 00:38:09,000
in the absence of growth factors
they won't divide.

420
00:38:09,000 --> 00:38:14,000
They'll just sit there.
But if we add PDGF or other growth

421
00:38:14,000 --> 00:38:19,000
factors, the cells will divide,
and therefore we'll see an increase

422
00:38:19,000 --> 00:38:24,000
in cell number.
So, again, the question is how does

423
00:38:24,000 --> 00:38:29,000
that happen?  What is the mechanism
that allows that to happen?

424
00:38:29,000 --> 00:38:43,000
Oops.  What did I do?

425
00:38:43,000 --> 00:38:48,000
Well, here we're talking about
another class of receptors.

426
00:38:48,000 --> 00:38:54,000
These are called growth factor
receptors.  And these are different

427
00:38:54,000 --> 00:38:59,000
from seven transmembrane receptors
in that they have,

428
00:38:59,000 --> 00:39:05,000
on their cytoplasmic side,
enzymatic activity, specifically

429
00:39:05,000 --> 00:39:10,000
kinase domains.
These receptors are also enzymes.

430
00:39:10,000 --> 00:39:16,000
And their enzymatic activity is to
add phosphates to other proteins.

431
00:39:16,000 --> 00:39:21,000
In this configuration, however,
they're inactive.

432
00:39:21,000 --> 00:39:27,000
What the growth factor does is
causes those receptors to dimerize

433
00:39:27,000 --> 00:39:33,000
with one another.
To become in close proximity to one

434
00:39:33,000 --> 00:39:39,000
another.  So the growth factor
serves to link them.

435
00:39:39,000 --> 00:39:46,000
Here's our growth factor.
And in this now increased proximity,

436
00:39:46,000 --> 00:39:53,000
these kinase domains become active.
And the consequence of that is we

437
00:39:53,000 --> 00:40:00,000
get phosphorylation of the
neighboring protein.

438
00:40:00,000 --> 00:40:11,000
We call this transphosphorylation.

439
00:40:11,000 --> 00:40:14,000
So ligand induced activation,
transphosphorylation.  And now what

440
00:40:14,000 --> 00:40:18,000
we've done, on the inside of the
cell, is to create a structure.

441
00:40:18,000 --> 00:40:22,000
And it's the formation of that
structure that then allows the rest

442
00:40:22,000 --> 00:40:51,000
of the process to work.

443
00:40:51,000 --> 00:40:56,000
These phosphorylated residues act as
the binding sites for other proteins.

444
00:40:56,000 --> 00:41:01,000
The first one is known
as an adaptor protein.

445
00:41:01,000 --> 00:41:10,000
An adaptor protein which binds to a

446
00:41:10,000 --> 00:41:16,000
class of proteins to that I referred
to previously,

447
00:41:16,000 --> 00:41:22,000
a guanine nucleotide exchange factor.
And that then allows the exchange

448
00:41:22,000 --> 00:41:28,000
factor to stimulate the exchange of
a small GTP binding protein,

449
00:41:28,000 --> 00:41:34,000
which is generally bound to GDP and
inactive, to stimulate the exchange

450
00:41:34,000 --> 00:41:40,000
of GTP for GDP.
This small GTP binding protein is a

451
00:41:40,000 --> 00:41:45,000
protein called RAS,
a very important signaling molecule.

452
00:41:45,000 --> 00:41:50,000
And, actually, a very important
cancer molecule as well.

453
00:41:50,000 --> 00:41:55,000
Mutationally activated in a high
percentage of cancers.

454
00:41:55,000 --> 00:42:00,000
When you now have RAS in its GTP
bound state, it can activate

455
00:42:00,000 --> 00:42:05,000
a kinase cascade.
Which I'll go through with you on

456
00:42:05,000 --> 00:42:11,000
slides in a moment.
It first activates a protein called

457
00:42:11,000 --> 00:42:16,000
RAF which then activates a protein
called MEK which then activates a

458
00:42:16,000 --> 00:42:21,000
protein called ERK.
ERK is a map kinase.

459
00:42:21,000 --> 00:42:27,000
It's a kinase itself.
Its enzymatic activity is to

460
00:42:27,000 --> 00:42:33,000
phosphorylate other proteins.
MEK phosphorylates ERK so it's

461
00:42:33,000 --> 00:42:40,000
called a map kinase kinase.
And RAF is a kinase itself which

462
00:42:40,000 --> 00:42:47,000
phosphorylates this kinase so it's
referred to as a map kinase kinase

463
00:42:47,000 --> 00:42:54,000
kinase.  I'm not making it up.
OK?  So it stimulates a kinase

464
00:42:54,000 --> 00:43:02,000
cascade, similar to what I
introduced you to before.

465
00:43:02,000 --> 00:43:06,000
And then, as you'll see,
ERK, which is a cytoplasmic protein,

466
00:43:06,000 --> 00:43:11,000
moves from the cytoplasm into the
nucleus.  I'll show this to you on

467
00:43:11,000 --> 00:43:16,000
slides, and it's in your book as
well.  And in the nucleus this

468
00:43:16,000 --> 00:43:21,000
kinase phosphorylates transcription
factors which then bind to target

469
00:43:21,000 --> 00:43:26,000
genes turning them on stimulating
the cell to divide.

470
00:43:26,000 --> 00:43:31,000
I would have drawn that for you but
we're running out of time.

471
00:43:31,000 --> 00:43:35,000
So I will show it to you instead on
the overheads.

472
00:43:35,000 --> 00:43:39,000
There's our wound.
So here's the pathway.

473
00:43:39,000 --> 00:43:43,000
It's just straight out of your book.
Here are the growth factor

474
00:43:43,000 --> 00:43:47,000
receptors, again,
not dimerized, not active.

475
00:43:47,000 --> 00:43:51,000
The growth factor causes the two
proteins to come together in close

476
00:43:51,000 --> 00:43:55,000
apposition.  This causes the kinase
activities to get stimulated

477
00:43:55,000 --> 00:44:00,000
resulting in the phosphorylation of
the cytoplasmic tails.

478
00:44:00,000 --> 00:44:04,000
The adaptor protein then can bind to
the phosphorylated tail.

479
00:44:04,000 --> 00:44:09,000
This then stimulates through an
exchange factor not shown here,

480
00:44:09,000 --> 00:44:14,000
the exchange of GDP for GTP, making
an active RAS molecule.

481
00:44:14,000 --> 00:44:19,000
This then can activate RAF making
it now an active kinase which

482
00:44:19,000 --> 00:44:24,000
activates MEK making it an active
kinase which activates ERK called

483
00:44:24,000 --> 00:44:28,000
map K here for map kinase.
This now goes into the nucleus of

484
00:44:28,000 --> 00:44:32,000
the cell from the cytoplasm,
and in the cytoplasm it can

485
00:44:32,000 --> 00:44:36,000
encounter transcription factors.
It then phosphorylates those

486
00:44:36,000 --> 00:44:40,000
transcription factors which bind to
their target genes turning on gene

487
00:44:40,000 --> 00:44:44,000
expression and leading to a change
in the cellular phenotype.

488
00:44:44,000 --> 00:44:48,000
And the change in the cellular
phenotype that we're talking about

489
00:44:48,000 --> 00:44:52,000
here is cell division,
the details of which we'll cover

490
00:44:52,000 --> 00:44:56,000
next time.  So this is sort
of a static picture.

491
00:44:56,000 --> 00:45:00,000
But to give you a more dynamic
picture of what's happening here,

492
00:45:00,000 --> 00:45:05,000
I borrowed from the Biocreations
Website an animation,

493
00:45:05,000 --> 00:45:10,000
which I find very helpful and,
actually, is kind of funny as well.

494
00:45:10,000 --> 00:45:15,000
So we're going to start this
process much the same way that we've

495
00:45:15,000 --> 00:45:20,000
just talked about using the terms
that I used on the board.

496
00:45:20,000 --> 00:45:25,000
Oop.  We need sound.  Let's do that
again so you can hear it

497
00:45:25,000 --> 00:45:29,000
in all its glory.  OK.
So the first thing is the receptors

498
00:45:29,000 --> 00:45:33,000
get dimerized by the growth factor
leading to the phosphorylation of

499
00:45:33,000 --> 00:45:37,000
these cytoplasmic tails.
And then this protein, which is the

500
00:45:37,000 --> 00:45:41,000
adaptor, it's called grab-2 grabs
onto one of those phosphorylation

501
00:45:41,000 --> 00:45:45,000
sites.  It has attached to it an
exchange factor called SAS here.

502
00:45:45,000 --> 00:45:49,000
OK?  So that's the first thing that
happens. You recruit this signaling

503
00:45:49,000 --> 00:45:53,000
complex to the membrane.
Once you have it there it's now in

504
00:45:53,000 --> 00:45:57,000
close proximity to RAS which is,
if you remember, a membrane

505
00:45:57,000 --> 00:46:01,000
associated protein.
It's now in its inactive stage,

506
00:46:01,000 --> 00:46:06,000
its GDP bound state.
Oops.  Sorry.  We'll just have to

507
00:46:06,000 --> 00:46:13,000
watch that again.
Oh, no.  Wait.  Wait.

508
00:46:13,000 --> 00:46:19,000
Wait.  Go back.  Forget you saw
that.  That's the end.

509
00:46:19,000 --> 00:46:26,000
You never want to see the ending.
Oh, wait a minute.  OK.  Here we go.

510
00:46:26,000 --> 00:46:32,000
OK.
So next we're going to activate RAS.

511
00:46:32,000 --> 00:46:36,000
It gets a little kick in the butt.
GDP comes off.

512
00:46:36,000 --> 00:46:41,000
GTP comes on.  Now RAS is active.
It's now capable of interacting

513
00:46:41,000 --> 00:46:46,000
productively with RAF,
the first of these kinases,

514
00:46:46,000 --> 00:46:50,000
the map kinase kinase kinase.
And it's going to cause it to

515
00:46:50,000 --> 00:46:55,000
become active.
Once it's active it's now capable

516
00:46:55,000 --> 00:47:00,000
of activating the next guy
in line, which is MEK.

517
00:47:00,000 --> 00:47:03,000
Now MEK is active,
and it's going to phosphorylate ERK,

518
00:47:03,000 --> 00:47:06,000
and now ERK is phosphorylated and
active.  Now, there have been a

519
00:47:06,000 --> 00:47:09,000
couple of other steps,
which you can look at, at home,

520
00:47:09,000 --> 00:47:12,000
but the RAS went from a GTP bound
stage to a GDP bound state.

521
00:47:12,000 --> 00:47:15,000
I told you that you can turn these
signals off up here.

522
00:47:15,000 --> 00:47:18,000
That happens in this pathway.
Another GAP comes along and turns

523
00:47:18,000 --> 00:47:21,000
RAS off.  And here's a protein
phosphatase, which is going to clip

524
00:47:21,000 --> 00:47:24,000
off the phosphate,
thereby turning MEK off.

525
00:47:24,000 --> 00:47:28,000
So each of these steps is
reversible.

526
00:47:28,000 --> 00:47:35,000
But, importantly,
we're down here at this level where

527
00:47:35,000 --> 00:47:42,000
ERK has been phosphorylated.
And now it's capable, dammit.

528
00:47:42,000 --> 00:47:49,000
All right.  We'll have to watch it
again.  Wait a minute.

529
00:47:49,000 --> 00:47:56,000
Come on.  OK.  Here we go.
I know.  It's like a video game.

530
00:47:56,000 --> 00:48:02,000
OK.
So ERK is active.

531
00:48:02,000 --> 00:48:06,000
And now it's going to go in the
nucleus.  Phosphorylate Jun,

532
00:48:06,000 --> 00:48:10,000
one of these transcription factors.
And upon doing so the transcription

533
00:48:10,000 --> 00:48:14,000
factors dimerize,
bind to the promoter of a target

534
00:48:14,000 --> 00:48:18,000
gene.  They recruit RNA polymerase.
And this leads to the production of

535
00:48:18,000 --> 00:48:22,000
new gene expression.
mRNAs that go into the cytoplasm

536
00:48:22,000 --> 00:48:26,000
get translated into proteins that
convince the cell now it's

537
00:48:26,000 --> 00:48:29,000
time to divide.  OK?
So I encourage you to look at this

538
00:48:29,000 --> 00:48:33,000
to get the details.
Now, if you just wait one more

539
00:48:33,000 --> 00:48:37,000
second.  In the last 30 seconds I
want to point out the fact that

540
00:48:37,000 --> 00:48:41,000
these pathways,
while we're teaching to you as

541
00:48:41,000 --> 00:48:44,000
simple linear pathways,
are actually highly complex.

542
00:48:44,000 --> 00:48:48,000
Pay attention to this clock here.
It's 11:54.  Highly complex, highly

543
00:48:48,000 --> 00:48:52,000
interactive, not linear,
and therefore we frequently

544
00:48:52,000 --> 00:48:56,000
analogize what's happening inside a
living cell to an integrated

545
00:48:56,000 --> 00:49:00,000
circuit.
And increasingly these days in

546
00:49:00,000 --> 00:49:04,000
biology, we're thinking about these
things in analogy with computers,

547
00:49:04,000 --> 00:49:09,000
and actually relying extensively on
computers to help us understand how

548
00:49:09,000 --> 00:49:13,000
that complexity actually works using
methods from chemistry and physics

549
00:49:13,000 --> 00:49:18,000
and mathematics and other
disciplines in a new and emerging

550
00:49:18,000 --> 00:49:22,000
area called systems biology,
which you might be interested in.

551
00:49:22,000 --> 00:49:27,000
And the new Biological Engineering
major will have a strong focus

552
00:49:27,000 --> 00:49:30,000
in this area.  OK.