BIOL 286 Chapter 16: Cell Communication C.

Mouzakitis
Extracellular signal molecules can act over short or long distances.
• Long distance: endocrine cell releases soluble hormone in to the blood. Hormone travels to distant
target cell. (analogy: radio)
• Local: paracrine cell releases hormone into extracellular matrix. Target cell is a short distance away.
(analogy: posting flyers in your neighborhood)
• Neuronal: though the axon can span a long distance (e.g., from spine to tip of toes), neurotransmitters
only travel a small distance in the synapse (100 nm, or two vesicle diameters), to target cell. (analogy:
sending a letter to a specific person)
• Contact-dependent: no soluble molecule released. Signaling cell has molecule on its surface, which
binds to receptor on the target cell. The two cells make contact for the transfer to occur. (analogy:
giving someone a message in-person)

All of these methods involve a signaling cell and a recipient cell.

The recipient cell will undergo a change in physiology upon receiving
the signal.

A signal is transferred to the cytoplasmic surface of receptor, the

signal is transmitted to an effector molecule, and there is an eventual
cessation of response.

Three main ways a receptor recognizes a stimulus:

1) Transmembrane receptor binds to ligand. Transmembrane
receptor changes its cytoplasmic tail, which brings the signal
inside the cell.
2) Transmembrane receptors bind to the extracellular matrix.
Instead of binding to a soluble molecule in the blood, the
transmembrane receptor binds to fibronectin or collagen protein.
3) Cell-Cell Contact: one cell binds to another. When cells touch
each other, they usually stop growing.

Signal transduction: converting one type of signal into

another. Extracellular signaling molecule (typically
hydrophilic) binds to cell and changes cytoplasmic tail of
transmembrane receptor, thereby inducing the activity of an
intracellular signaling molecule.

First messenger: hydrophilic molecule outside the cell.

Example: soluble hormone diffusing through the blood.

Effector: releases soluble secondary messengers within the

cell.

Secondary messenger: molecule inside the cell that binds to

other molecules to change their shape and alter their
activity.
Amplification: One first messenger will be transduced to
activate multiple second messengers inside the cell.
Integration: signals from more than one intracellular
signaling pathway are detected and integrated before the
signal is relayed. Multiple signal pathways typically
converge on one highly modified protein.
18:20 in Lecture 15; pages 534-535 in textbook

Distribution: the distribution of one signal to more than one effector protein, creating branches in the
information flow diagram and invoking a complex response. One protein is essentially responsible for changing
several things about the cell.
The same signal molecule can induce
different responses in different target cells.

Shown left, acetylcholine binds to an

acetylcholine receptor in both a heart
pacemaker cell and a salivary gland cell. In
the pacemaker cell, acetylcholine will slow
the rate of firing. In the salivary cell,
acetylcholine will induce the secretion of
amylases.

The two cells have the same receptor, but

different intracellular effector proteins are
activated.
In other cases, the same signaling molecule will bind to a different receptor to induce different changes. In the
skeletal muscle cell, acetylcholine will bind to a different receptor to open sodium channels and cause muscle
contraction.

Though most signaling molecules are hydrophilic, cannot cross the

bilayer, and therefore remain on the cell surface, there are some signaling
molecules that are hydrophobic. A carrier protein protects these small
hydrophobic molecules as they travel through the blood. They are
released from this carrier at the surface of the cell, and they can readily
diffuse through the membrane. They will bind to an intracellular receptor
in the cytoplasm. Example: steroid signaling.

Animal cells have multiple receptors and receive multiple signals. A

healthy cell receives signals telling it to stay alive. It will receive additional signals telling it to grow and
divide. These second signals will stop, and a new set of signals will tell the new cells to differentiate
themselves (cells continue receiving survival signals). Cells that lack signals usually die.

Extracellular signals can act rapidly or slowly.

If the extracellular signal molecule leads to a pathway that

alters protein function (e.g., via phosphorylation of the
protein), changes in the cell can be very rapid. Example:
vision.

If the extracellular signal molecule leads to a pathway that

alters gene expression (e.g., activation of a transcription
factor to produce RNA to be translated into a new protein),
changes in the cell are very slow.

In both cases, the signal eventually shuts off, whether it is by

dephosphorylating the protein (in the first situation) or by degrading the produced protein (in the second
situation).
Both are signal pathways: series of distinct proteins that alter the conformation of the “downstream” protein.
Second Messengers: a first messenger will bind to a transmembrane receptor, which causes an intracellular
protein to release a second messenger. These second messengers are small, diffusible nonprotein intermediaries
acting in signal transduction. Examples: cAMP, calcium, lipid-derived.

3 Classes of Transmembrane Cell Surface Receptors

1. Ion channel coupled receptors
Example: acetylcholine receptor
Initially, the channel is closed, but the binding of
acetylcholine (first messenger) will open the channel to
allow the passage of ions (second messenger) into the
cell. First messenger stays outside the cell.

2. G-protein-coupled receptors
The binding of a signaling molecule to its
transmembrane receptor will cause a shape change
allowing the receptor to bind to the G protein. The
bound G protein will be activated via a shape
change, giving it an affinity for a nearby enzyme
(effector). The enzyme will now relay the signal
inside the cell.

3. Enzyme-coupled receptors
The transmembrane receptor itself can
be an enzyme. It begins as two
monomers, which dimerize when a
signaling molecule is bound. The two
monomers become kinases that
phosphorylate each other. This activates
each monomer, creating an active
catalytic domain. If the transmembrane
receptor itself is not an enzyme, dimerization via the binding of a signaling molecule will activate an associated
enzyme to carry out changes in the cell.
37:50 in Lecture 15

Characteristics of cell-signaling systems: many intracellular signaling molecules act as “molecular switches”
Two major ways a protein can undergo a shape change:
1. Protein Phosphorylation (RAPID)
• Phosphate is covalently added to an amino acid, which causes a
conformational change in the entire protein.
• Shape change may be part of the protein’s binding site.
• Amino acids with OH groups in their side chain (e.g., serine,
tyrosine, or threonine) are capable of being phosphorylated.
• Extracellular signal will activate a protein kinase, which removes
the gamma phosphate from ATP and places it on the target protein.
• Protein phosphatase can break the covalent bond on the
phosphorylated protein and return the protein to its original state.
• Phosphorylation may increase or decrease a protein’s activity;
phosphorylation may cause the protein to change from OFF to ON,
or from ON to OFF.
• The kinase and phosphatase themselves require regulation via
phosphorylation. Phosphorylation will active them so that they may
phosphorylate other proteins.
 Shown left is a signaling pathway that will activate a transcription
factor to bind to DNA and turn on a gene.
Most substrates are other enzymes themselves. The target of a
kinase is often another kinase.
Protein kinase 1 may be activated because a receptor binds to a
ligand. It will phosphorylate protein kinase 2, thereby activating it.
Protein kinase 2 will then phosphorylate protein kinase 3.
Evidently, phosphorylated proteins are also kinases. Protein kinase
3 will then phosphorylate the transcription factor to activate it.
Protein kinases/phosphatases change the shape/activities of the
proteins they modify.
Continual balance of kinase and phosphatase activities. Whichever
enzyme is more active at the time will be the net result.
Human genome has ~500 different kinases and ~100 different
phosphates. Genome has a lot of space utilizing kinases.

The advantage of having all the middle kinases is that each can be regulated. For instance, protein kinase 1
may phosphorylate protein kinase 2, but a hormone pathway may also phosphorylate protein kinase 2. Thus,
there is integration of multiple signals on each kinase.

2. GTP-Binding Proteins
• G-proteins are not kinases. They are bound to GDP at one
shape, and GTP at another shape. There is a shape change
by GDP/GTP exchange.
• Unlike a kinase, which can turn a protein on or off via
phosphorylation, GDP-bound proteins are always off and
GTP-bound proteins are always on.
• A G protein is initially bound to GDP. A signal will cause
the GDP to be removed and a GTP to come in. There is no
phosphorylation here (GDP does not become GTP).
However, the released GDP (no longer associated with a G
protein) can have phosphate added to it to become GTP.
• There is more GTP in the cytoplasm.
• This is not a covalent modification. There is a specific
binding site on the G protein for GDP or GTP.
• The G protein can hydrolyze itself to return to the GDP-bound form, thus turning itself off.
• The target of a GTP protein does not become phosphorylated (like the kinase phosphorylation
pathways). GTP-bound proteins bind to their target and change it.

Activity of monomeric GTP-binding proteins is controlled by two types of regulatory proteins (shown above):
A) GEF: Since GDP is tightly bound, guanine nucleotide-exchange factors bind to a G protein, causing it
to change its shape and release GDP. This allows GTP to bind.
B) GAP: Though the GTP-bound protein can hydrolyze itself to return to GDP, this process is very slow.
GTPase-activating proteins speed up this hydrolysis by 18,000 fold.

52:20 in Lecture 15
 Two Alternate types of Signal Transduction Pathways
A) G protein-linked receptors: pathway that begins with the left
transmembrane receptor. Via a G protein, a transmembrane receptor will
activate an effector, which will lead to a series of events within the cell.
B) Protein kinase receptor: pathway that begins with the right
transmembrane receptor. Discussed later in the outline.

G protein-linked receptors or G protein

coupled receptors (GPCRs) have a similar
structure: 7 transmembrane α-helices. An
external face binds to a signal, and a
cytoplasmic tail which changes its shape.
One face will have an amino terminus, and
the other will have a carboxy terminus.
They may have very different structures, but
they all cross the membrane 7 times.
Shown left is a G protein coupled receptor. A
receptor is linked to an effector by a G protein.
GPCRs are the largest superfamily of proteins in
animal genome (e.g., nematode has 19,000 genes of
which 1,000 are GPCRs).
GPCRs are the target of ~40% of medicinal drugs.
Glucagon receptor activates a G protein, which
activates the adenylyl cyclase effector, which will
make and release the cAMP second messenger.

The G protein shown above is heterotrimeric: it is made of three different molecules. The α and γ subunits
each have a lipid tail covalently linked to the bilayer. The β and γ subunits are usually linked. The α subunit
has a binding site for GDP or GTP.

G-protein activation (shown right):

A heterotrimeric G protein is shown bound to GDP. It has no affinity
for the receptor, as the receptor is not bound to a signal molecule yet.

When the G protein coupled receptor’s extracellular face binds to its

ligand (signal molecule), the receptor’s intracellular tail will change its
shape. The receptor now has an affinity for the α subunit of the G
protein.

When the receptor and the G protein’s α subunit bind, GDP leaves.
The activated receptor in this case acts as a guanine nucleotide
exchange factor (GEF).

GTP will bind to and activate the α subunit of the G protein. This will
release the βγ subunit, which is also activated. Either the α or βγ
subunits can now bind to different effectors.

1:00:00 in Lecture 15
 The GTP-bound α subunit of the G protein can diffuse in the
plane of the membrane since it has a lipid tail. It binds to a
target molecule (effector), thus changing the shape and
activity of the target molecule.

Ending the Response: turning off the effector (shown left)

The G protein will hydrolyze GTP back to GDP. In the GDP
form, it will no longer have an affinity for the target molecule
(effector), and will therefore dissociate. The dissociated
effector is now off.

Now that the α subunit of the G protein is in the GDP form, it

has an affinity for the βγ subunit. The trimer is reformed.

Note that the α subunit does not really rebind to its original
βγ subunit. The binding of a ligand to a receptor will cause
many G proteins to become active, which means there are
many βγ subunits diffusing in the plane of the membrane.

Ending the response: receptor desensitization (shown above)

1. GRK (G protein-coupled receptor kinase): phosphorylates the cytoplasmic tail of a receptor. (3 P)
2. Arrestin: recognizes the phosphorylated tail of the receptor and binds to the tail. This blocks G protein
binding. Despite having a ligand bound on the extracellular face, and having a tail in the proper shape
for G protein activation, the receptor will be unable to activate G proteins because it is blocked.
3. The receptor can also be endocytosed from the plasma membrane when arrestin binds to clathrin
(vesicle formation). It is taken into the cytoplasm such that it can no longer bind to extracellular signal
molecules. Receptors can be degraded in lysosomes at this step.
Signals can also be inhibited if the ligand is degraded. Example: there are enzymes outside the cell that degrade
acetylcholine and remove it from the receptor’s binding site.
Video on G-protein signaling shown in lecture: https://www.youtube.com/watch?v=V_0EcUr_txk

Upon prolonged stimulation of G proteins, a receptor will eventually inactivate, even if its ligand is still bound.

Specificity of G protein-coupled responses:

• Not all parts of signal transduction machinery identical in all cells
• Multiple forms of receptors (e.g., 9 different isoforms of epinephrine receptors) with different ligand
and G protein affinities. Example: different epinephrine levels will trigger a response from different
receptors (concentration of external ligand determines receptor response).
• Multiple G proteins (20 Gα, 5 Gβ, 11 Gγ); various combinations give different specificities.
• Gα s: Some G protein α subunits will stimulate an effector when activated.
• Gα i: Some G protein α subunits will inhibit an effector when activated (second messenger decreased).
8:10 in Lecture 16
Some G proteins directly regulate ion channels.
Example: slowing the heart rate with
acetylcholine
Acetylcholine binds to a receptor on a heart cell.
This activates the receptor, which causes a G
protein to exchange its GDP for GTP.
The G protein will release its activated βγ subunit.
The dissociated βγ subunit can now bind to an ion
channel, not because of a shape change, but
because dissociation from the α subunit frees up
the site that the βγ subunit uses to bind to the ion
channel.
When the βγ subunit binds to the cytoplasmic tail
of the ligand-gated ion channel, it causes the
channel to open. Potassium leaves the cell, making the inside of the cell even more negative. This
hyperpolarization slows the heart rate. When channel closes, Na+/K+ pump will restore intracellular K+ levels.

Some G proteins activate membrane-bound enzymes (the effectors in the signal pathway).
• Adenyl cyclase: increases cAMP
• Phospholipase C: cuts lipids

Glycogen (excess glucose) is stored

in the liver. When the body is
exerted, it will break down glycogen
into usable glucose.

Glycogen phosphorylase – makes

more glucose – uses inorganic
phosphate to break the bonds in
glycogen and generate glucose 1-
phosphate. The glucose 1-phosphate
becomes glucose 6-phosphate, which
can enter two pathways. The first pathway is glycolysis (especially in muscle cells, which use glucose quickly),
and the second is the release of glucose into the blood (especially in liver).

Glycogen synthase – makes more glycogen – extra glucose will trigger the release of the insulin hormone,
which initiates the activity of glycogen synthase. Glucose 1-phosphates in the blood are added onto glycogen
chains.

Glucagon is released from the pancreas and binds to the surface of liver cells, leading to the breakdown of
glycogen and release of glucose. Epinephrine is released from the adrenal gland and binds to different
receptors on liver cells. The two hormones work to increase blood glucose not only by activating
phosphorylase, but also by inhibiting glycogen synthase.

The two hormones bind to different receptors, yet lead to the same intracellular response. This is because they
both use the same secondary messenger cAMP. They have different receptors and different G proteins, but
the same cAMP secondary messenger.

Note: epinephrine can also bind to heart cells to increase heart rate. Evidently, depending on the type of
receptor, a signal molecule can have drastically different effects on cells.
Adenyl cyclase (effector): an integral membrane protein with 12
transmembrane domains. It uses ATP to make cAMP. Glucagon or
epinephrine will bind to a receptor, which activates a G protein, which
activates the adenyl cyclase to make more cAMP.
cAMP diffuses through the cell to activate a kinase involved in glycogen
breakdown.

First messenger: glucagon/epinephrine outside the cell

Second messenger: cAMP inside the cell

Adrenaline stimulates glycogen breakdown in skeletal

muscle cells:

Shown left, adrenaline and glucagon have different

receptors and G proteins. When bound to a receptor,
they both activate adenylyl cyclase, which releases
cAMP. As a second messenger, cAMP will diffuse
through the cell and bind to inactive protein kinase A
(PKA) in the cytoplasm. Active PKA will
phosphorylate inactive phosphorylase kinase. Once
phosphorylase kinase becomes activated, it will
phosphorylate glycogen phosphorylase, which will
begin breaking glycogen.

Multiple steps allow the cell to fine tune its response

with more opportunities for regulation and
amplification.

23:53 in Lecture 16

Shown right, in addition to breaking glycogen, the cell

will also block the synthesis of new glycogen. cAMP
will activate PKA (as discussed above), which can
phosphorylate glycogen synthase. Unlike glycogen
phosphorylase, which becomes active when
phosphorylated, glycogen synthase becomes inactive
when phosphorylated. Glycogen synthesis is inhibited.

PKA has a third function: the activation of gene transcription. The cell
can increase blood glucose by activating a transcription factor that will
initiate the production of enzymes needed for gluconeogenesis. These
enzymes can use glycerol, lactate, or certain amino acids to generate
glucose. Activated PKA (via release of cAMP) can enter the nucleus
through a nuclear pore and phosphorylate an inactive transcription factor.
The active transcription factor will bind upstream in the gene regulatory
region to activate the target gene. The mRNA produced can translate to
protein enzymes.
Video on cAMP signaling shown in lecture:
http://www.youtube.com/watch?v=iGb93jCKVXs
Having multiple steps in these pathways allows for signal amplification.
• Each G protein coupled receptor (GPCR) activates multiple G proteins as
long as its ligand is bound. Note that the G proteins in one pathway are not
different; they are multiple copies of the same αβγ subunits
• Each G protein can activate multiple adenylyl cyclases
• Each active adenylyl cyclase makes many cAMPs
• Each cAMP can activate many PKAs
• Each active PKA can phosphorylate multiple phosphorylase kinases
• Glycogen breakdown becomes quite rapid
There is further regulation in that the phosphate added to phosphorylase by PKA
can be removed by another phosphorylase, which can also be regulated by a different pathway.
Reversal of signal
• cAMP can be destroyed by cAMP phosphodiesterase, which converts cAMP to AMP. The destroyed
cAMP will no longer activate PKA.
• Phosphatase-1 removes the phosphates that were added by PKA onto phosphorylase kinase. It can also
dephosphorylate active glycogen phosphorylates.
• cAMP phosphodiesterase and phosphatase-1 themselves are regulated by other pathways.

Another effector that is activated by a G protein coupled receptor is

phospholipase C (the first was adenylyl cyclase). Its activation will
release IP3 and DAG as second messengers throughout the cell
(adenylyl cyclase released cAMP).
Shown left is a phosphoglyceride called phosphatidylinositol.
Recall, phosphoglycerides have a glycerol backbone, two fatty acid
tails, a phosphate, and a head group.
A phospholipase is a hydrolytic enzyme that splits phospholipids.
IP3 DAG Phospholipases A, D, and C (PLA, PLD, and PLC) each cut at
specific sites on the phospholipid.

Phosphatidylinositol is best studied as its derivatives. Phospholipase C will cleave phosphatidylinositol along
the dotted line. This yields diacylglycerol (DAG), which is two fatty acid tails embedded in the plasma
membrane and a glycerol backbone, and IP3, which is the phosphate and the inositol (which has two phosphates
of its own). Note that before IP3 is produced, that section of the phosphatidylinositol is called PIP2.

A ligand will bind to a G protein coupled receptor, which will activate a G protein, which will activate
Phospholipase C. Phospholipase C (PLC) will then cleave phosphatidylinositol to yield DAG (remains in
membrane) and IP3.
41:26 in Lecture 16

Diacylglycerol (DAG):
• Lipid molecule that remains in the plasma membrane after cleavage from phosphatidylinositol
• Readily diffuses in the plane of the membrane
• Will recruit and activate protein kinase C (PKC)
• PKC is a serine/threonine kinase (two of the three amino acids that can be phosphorylated). This
means it will phosphorylate serines and threonines in its target protein. It is important in many cellular
events (e.g., cell growth, differentiation, metabolism, and transcriptional activation)
• We study DAG using phorbol esters, which are plant compounds that mimic DAG. This allows us to
trick cells into activating PKC without any signaling molecules (bypassing receptor part of the
pathway). This continuous bombardment with phorbol esters will cause cell to lose growth control and
behave as a malignant cell.
IP3 (inositol 1,4,5-triphosphate):
• Small, water soluble, hydrophilic
• Binds to a Ca++ channel receptor on the smooth endoplasmic reticulum (SER)
• Calcium that the SER sequestered is then released into the cytoplasm

A signaling molecule will activate a G protein

coupled receptor. The alpha subunit changes GDP
for GTP. The active alpha subunit activates
phospholipase C, which cleaves
phosphatidylinositol, releasing IP3 and DAG. DAG
activates PKC, which leads to cell growth, division,
and differentiation. IP3 binds to receptors on the
SER (binds to a ligand-gated channel). Calcium
leaves the ER lumen and enters the cytoplasm. An
increase in calcium levels in the cytoplasm (where
levels are usually very low) will stimulate other
responses. For instance, DAG activates PKC, but
PKC also requires calcium to function.

Calcium as an intracellular messenger:

• Ca++ release from intracellular stores acts as a second messenger (like cAMP and DAG/IP3). Do not
call calcium a third messenger even though it seems more appropriate.
• Recall, a first messenger could be a hormone, neurotransmitter, electrical activation (muscle) on the
outside of the cell.
Video on calcium signaling shown in lecture: http://www.youtube.com/watch?v=jWhc59XWPSU

From video: Occasionally, calcium waves are transmitted to adjacent cells through gap junctions.

Ca++ is not made enzymatically. Unlike cAMP, which is made from ATP, and IP3, which is made from PIP2
cleavage from phosphatidylinositol, calcium is just stored in the ER lumen until it is required and then returned
to the ER lumen. It is not synthesized or degraded.
• [Ca++] controlled by pumps and channels
• Free intracellular [Ca++] is ~0.1 µM
• [Ca++] within lumen of ER is ~1 mM
• Concentration is 10,000-fold higher in ER lumen and outside of cell compared to inside cytoplasm
• Channels into cytoplasm are normally closed
• Pumps bringing calcium outside of the cytoplasm are usually active

Ca++ release channels are found on the surface of the smooth ER, where
IP3 receptors (IP3Rs) are also found. Degradation of the IP3 will close the
channels.

While cAMP acts on kinases, primarily PKA, Ca++ modulates a broad

range of effectors. Ca++ can activate kinases (PKC), ion pumps, proteases,
phosphatases, Ca++-binding proteins, etc.

Calmodulin is a protein that senses calcium levels and changes its shape
when levels are high. It is the best known Ca++-binding protein.
 Calmodulin is unfolded (far left) when it is not bound to
calcium. It binds to calcium only in stimulated cells
(affinity is too low). It has four calcium binding sites.

Calcium binding will cause a change in conformation and

affinity for other proteins. As shown, bound calmodulin
will bind to a target protein to relay the signal.

Video on calmodulin shown in lecture:

http://www.youtube.com/watch?v=sufhrXpuoF8

57:20 in Lecture 16

Role of GPCR in vision: a response is 20 milliseconds

(20/1000th of a second)
• Ligand: light (photon)
• Receptor: rhodopsin
• G protein: transducin
• Effector: cGMP phosphodiesterase

Guanylate cyclase synthesizes cGMP from GTP. cGMP levels are

kept very high, which keeps sodium channels open. The activation
of the effector cGMP phosphodiesterase will begin breaking this
cGMP, causing sodium channels to close.

Shown right is a rod cell that has inactive rhodopsin in the dark. Cell
has its sodium channels open (resting potential -30 mV, which is
higher than regular neurons). Sodium is continuously flowing into the rod cell (as is calcium), causing the
release of a high amount of neurotransmitters. When light hits the rod cell, sodium channels are closed, and the
cell becomes hyperpolarized. Release of neurotransmitters (in vesicles) is
blocked.

Amplification:
Shown left, binding of one photon to one rhodopsin molecule will activate
500 G-proteins (transducin). Each G protein will activate a cGMP
phosphodiesterase. Each cGMP phosphodiesterase can hydrolyze 105
cGMP molecules. This will close 250 sodium channels.

106-107 sodium ions per second are prevented from entering the cell for a
period of ~1 second. This alters membrane potential by 1 mV. In the
dark, sodium is continuously leaking into the cell. In light, sodium
channels are closed.

Rhodopsin eventually becomes phosphorylated and inactivated. This

entire process takes about 50 milliseconds (very rapid).

On page 5, I wrote that protein kinase receptors, the second type of signal transduction pathway (first was G
protein coupled receptors) would be discussed later in the outline. The final cell-signaling lecture deals
exclusively with protein kinase receptors.
Protein Kinase Receptors are also called Receptor Tyrosine Kinases (RTKs)
• Transmembrane receptor
• These kinase enzymes will
add phosphate groups to
tyrosine amino acids on
other proteins
• Each RTK monomer
crosses plasma membrane
once (recall, GPCRs cross 7
times)
• Over 50 different RTKs
have been identified
As shown above, inactive RTKs begin as two monomers separated in the plasma membrane.
• Ligand binding induces dimerization of the two monomers, as well as autophosphorylation. Ligand is
considered a bridge between the two monomers.
• The tyrosine residues that are now phosphorylated become binding sites for signaling proteins that were
originally floating in the cytoplasm.
• Binding of signaling protein to a phosphorylated tyrosine on an RTK will activate that signaling protein.
9:05 in Lecture 17

Phosphotyrosine motifs: only tyrosines surrounded by certain amino acids are

phosphorylated. Not all tyrosines on the receptor are phosphorylated.
Phosphorylated tyrosines become docking sites for specific signaling proteins.

SH2 domains (src homology 2 domains) and PTB domains (phosphotyrosine binding domains):
• A domain is a region of a protein that originated from a single exon and can fold on its own
• SH2 domains are similar regions on different molecules that can recognize and bind to phosphotyrosines
• PTB domains have the same function as SH2 domains
• Even proteins with very different functions that have an SH2 domain will be directed to a specific
phosphotyrosine on the RTK.
• SH2 structure is a compact “plug-in” module that can be inserted nearly anywhere in a protein sequence
without disturbing protein folding

Role of RTK in cellular activities

• Hormones – insulin and growth hormones can bind to RTKs
• Growth factors – epidermal growth factors (EGF), fibroblast growth factor (FGF), platelet-derived
growth factor (PDGF) can bind to RTKs causing a cell to divide and grow

Insulin Receptor (Glucose uptake)

After a large meal, pancreatic cells sense the increased
glucose levels. They will release insulin throughout the
body. Insulin will bind to receptors on cells (e.g., in
liver) causing the cells to take up the glucose and store
it as glycogen.
• Tetramer of two α and two β polypeptide chains
• Technically one molecule since the subunit are
linked with disulfide bridges
• Insulin binding to α (extracellular) changes the conformation of β (intracellular)
• Activated receptor phosphorylates itself (autophosphorylation)
• Soluble proteins (insulin receptor substrates: IRSs) will use their PTB domains to bind to the
phosphotyrosine
 PTB domain of an IRS-1 protein will bind to the phosphorylated insulin receptor

The receptor will phosphorylate tyrosines on the IRS-1 protein, whose phosphotyrosines will
then serve as docking sites for other signaling proteins.

The IRS-1 protein is shown right.

It includes a PTB domain that
binds to the insulin receptor, as
well as three docking sites (in brackets) for inactive proteins floating in the cytoplasm. The
three docking sites will active three different pathways. This means that insulin binding can initiate several
processes in the cell.

IRS-1 protein uses a PTB domain to bind to the insulin receptor. A docking
protein on IRS-1 called Grb2 will use an SH2 domain to bind to a
phosphotyrosine on IRS-1.

Two docking proteins that are activated by IRS-1 are Grb2, as part of the
Ras pathway, and PI-3-K (PI-3-Kinase).
Can continue to make
21:07 in Lecture 17 PI(3, 4, and 5)

Note that there are cases in which PI-3-K can bind directly to an RTK.
It does not require IRS-1 for docking.

Activated PI-3-Kinase will phosphorylate phosphatidylinositol. As

shown left, the same kinase can add phosphates again and again to the
same inositol ring. The newly made phosphoinositides can direct
major signaling cascades by acting as docking sites and as secondary
messengers.

The phosphorylated inositol rings bind to modules on many proteins. They recruit specific proteins to the
cytoplasmic face of the plasma membrane.
The signaling molecules that bind to the phosphorylated inositol rings do not have PTB or SH2 domains.
Instead, they have PH (pleckstrin homology) domains. These domains were first identified in platelet
pleckstrin proteins, but have since been found in other proteins. They target proteins to membranes by binding
PIP2 or PIP3 when they are produced.

GFP (green fluorescence protein) was fused to a PH. We expect that it will bind to PIP3, since PH is a binding
domain for PIP3. After the growth factor PDGF is added to the cell, PIP3 is produced and the GFP fused to PH
will migrate to the cytoplasmic face of the membrane and bind to PIP3.

Newly formed PIP3 now serves as docking site for PKB (Protein Kinase B also
called Akt). PKB binding to PIP3 only partially activates PKB. PKB also
requires phosphorylation by protein kinase 1, another kinase with a PH domain,
and protein kinase 2, for full activation.

32:10 in Lecture 17
 PKB (shown in brown) binds to and is partially activated by PIP2 and
PIP3 via its PH domains. It is then fully activated by protein kinase 1
(shown in blue).

Fully activated PKB will lead to glucose uptake (our original goal
when insulin fused to the RTK on a cell in the liver), glycogen
synthesis (activation of glycogen synthase), and protein synthesis.
These three activities lead to lower blood glucose.

Signal is reversed by dephosphorylating the insulin receptor.

Glucose Uptake Pathway

Insulin receptor will bind insulin. IRS-1 will dock. PI-3-Kinase is
activated. PKB is activated. Glucose transporters, which allow glucose to
diffuse down its concentration gradient, are inactive when on a cytoplasmic
vesicle. PKB activation will place them back into the membrane via fusion
with plasma membrane. Glucose uptake into the liver cell allows it to be
stored as glycogen.

Insulin receptors and growth factor receptors are also involved in the Ras pathway
• Ras is a monomeric G protein held to plasma membrane by lipid group
• Monomeric G proteins mimic Gα in function
• Nearly all RTK activate Ras
• Since ras stimulates cells to divide, 30% of human tumors have a mutated ras gene, causing the ras
protein to remain in the GTP form (GTP hydrolysis is prevented) and force the cell to divide
continuously. Ras is always “on.”

Ras (shown in green) has a lipid tail

keeping it in the membrane. A
growth factor or insulin will bind to
RTK. RTK will autophosphorylate
and bind to Grb2 and Sos. These
two proteins will cause Ras to
exchange its GDP for GTP, thereby
activating Ras protein.
Sas acts as a guanine nucleotide
exchange factor (GEF), which
catalyzes this GDP-GTP exchange.
Video on Ras shown in lecture: https://www.youtube.com/watch?v=TA_2WbGA0zw

Active Ras protein will activate MAP kinase pathway (Mitogen-Activated Protein Kinase).
• Growth factor binding leads to increased transcription of genes involved in growth responses
• Mitogen induces mitosis of the cell
• Ras GTP hydrolysis is induced by a GTPase activating protein (30 min without enzyme; 1/10 of a
second with enzyme)

46:10 in Lecture 17
 Note the box in the image on the left. Kinases are named after what
they phosphorylate. Working backwards, a transcription factor is
phosphorylated by MAPK. In order for MAPK to activate, it must
be phosphorylated by MAPKK. MAPKK requires phosphorylation
by MAPKKK to activate. For simplification, these kinases are
called ERK, MEK, and Raf, respectively.

Exam Question: Ras-GTP IS NOT A KINASE. G proteins are not

kinases. Ras-GTP will bind to the first kinase (MAPKKK), which
begins the Ras kinase pathway. Though it looks as though Ras-GTP
is phosphorylating Raf, there are many intermediate steps before this
phosphorylation. Ras-GTP does not do the phosphorylation itself.

The phosphorylated
transcription factor can
change gene expression
or protein activity. The
MKP-1 shown is a
phosphatase that turns
off the upstream
pathway.

On page one, we said there are three ways a receptor

recognizes a stimulus. We have been discussing the
binding of a ligand to a receptor. Now we will discuss
the binding of the extracellular matrix to a receptor. This
binding can also activate the Ras pathway.

Signals that originate from the Extracellular Matrix

• Integrins transmit signals that influence cell
growth, shape, migration, differentiation, and
survival
• Normal cells do not grow in suspension. This
means that normal cells found in media like blood
are not growing. This is because they are not
bound to an extracellular matrix.
• Transformed cells (e.g., cancer cells) grow
constantly without being attached to a matrix.

Integrin binding to an extracellular matrix will change its

cytoplasmic tail. This allows several proteins to bind to
its tail. These proteins will eventually active the Src
protein.
 1) Clustering of integrins induces protein binding
2) Phosphotyrosin activation of src. Src is an oncogene, which means it is a protein kinase in a cell that,
when mutated, will cause the cell to lose growth control and become cancerous.
3) Phosphotyrosin activation of FAK kinases. FAK (Focal adhesion kinase) will activate Grb2 and Sos.
4) Ras activated
5) MAP kinase cascade increases transcription of genes involved in growth

At the tails of the integrins, actin filaments are found (shown in green). Phosphotyrosine
proteins are shown in orange. Concentrations are very high at these FAK hot spots.

Highest concentration of phosphotyrosine in a cell is at the ends of the actin bundles.

58:40 in Lecture 17

Many signaling ligands (and the extracellular matrix) are hydrophilic and cannot cross the lipid bilayer.
Receptors for these ligands are transmembrane.

When a signaling molecule is hydrophobic, it can cross the bilayer and bind to receptors inside the cytoplasm.
Its crossing into the cell is aided by a carrier protein.

Role of Nitric Oxide (not laughing gas) as an intracellular messenger involved in smooth muscle relaxation

Acetylcholine receptors are found on the surface of the endothelial cells. Binding of
acetylcholine will cause the cell to convert arginine into nitric oxide with the help of
nitric oxide synthatase.

Nitric oxide diffuses through the lipid bilayer and enters the smooth muscle cells around
the endothelial cells it was originally in. The nitric oxide binds to a receptor (guanylyl
cyclase) in the cytoplasm of the smooth muscle cell. This converts GTP to cGMP.
cGMP will relax the smooth muscle.

Mode of Action for Viagra

• Nitric Oxide (NO) released by nerve and endothelial cells in penis
• NO à higher cGMP levels à muscle relaxation and increased flood flow à erection
• The cGMP is eventually degraded by phosphodiesterase. Viagra will inhibit the activity of this
phosphodiesterase (PDE5). It is a unique form of cGMP phosphodiesterase found only in the penis.
Viagra might inhibit a phosphodiesterase involved in vision, which is why some people on Viagra see blue.
Cell death occurs when a cell no longer receives signals. Cell can also receive a signal to die.

Programmed Cell Death (Apoptosis)

• “Cell suicide” occurs during development
• In adults, apoptosis removes unnecessary cells (e.g., immune cells after infection gone)
• Main defense against cancer development
• Signal activation phase followed by execution phase
 Cell death slides will be discussed further in a later lecture
Execution phase is best understood:

• Cytoplasm and nucleus shrinks

• Cell rounds up and loses cell-cell contacts
• DNA fragments; cell may also fragment
• Phosphatidylserine on outer plasma membrane leaflet are the “eat me” signals for phagocytic cells
Signal Activation Phase
• External signals can activate plasma membrane receptors
• Internal damage signals can activate via mitochondria
• Caspases: proteases that induce changes during execution phase. They can proteolyze:
o Protein kinases (e.g., FAK, PKA, PKC)
o Nuclear envelope proteins (lamins)
o Cell structure proteins (e.g., actin, IF)
o DNA repair enzymes
o DNase inhibitory protein (now cleaves DNA)

Signaling cascades are not just linear:

Convergence: 2 ligands, 1 path

Divergence: 1 ligand, many paths

Scaffolds help organize these cascades.

Scaffolds are structure—not enzymatic—
components of the pathways. They provide
spatial localization, by physically separating
kinases, and substrate specificity.