SIGNAL

TRANSDUCTION
David Henkin
Cell signalling the general
Glucocorticoid / Thyroid hormone
signalling
4
The three largest classes of cell-surface receptor
proteins:
Ion-channeled coupled receptors
Mediated by NT
Therefore involved in synaptic signalling
G-protein-coupled receptors
(also called Heptahelical receptors)
Act by indirectly regulating the activity of a separate plasma-membrane-
bound target protein, which is generally either an enzyme or an ion channel
A trimeric GTP-binding protein mediates the interaction between the
activated receptor and this target protein
Enzyme-coupled receptors
Either function directly as enzymes or associate directly with enzymes that
they activate
Examples
Tyrosine kinase receptors
Serine/threonine kinase receptors
G-proteins have this general structure
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Intracellular effects of the cAMP pathway:
Control of glycogen degradation
Epinephrine binds b-Adrenergic receptor
Gs protein binds GTP, dissociates from other subunits
Activated Gs binds and activates adenylate cyclase
Adenylate cyclase produces cAMP from ATP
PKA binds cAMP and becomes activated
PKA phosphorylates and activates Phosphorylase Kinase
Active Phosphorylase Kinase phosphorylates phosphorylase b
into the active phosphorylase a form
Phosphorylase a catalyzes the phosphorolytic cleavage of
glycogen into glucose-1-phosphate
Beta-adrenergic
receptor = a G-protein
coupled receptor
Simultaneously, PKA also
phosphorylates glycogen
synthase, this shuts
down glycogen synthesis
Regulation of glycogen metabolism by Gs
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GTP
bound
PKA phosphorylates
Phosphorylated to turn off:
Glycogen Synthase
Phosphorylated to turn on:
Phosphorylase Kinase
Phosphorylase B A
PKA phosphorylates TFs
***Gs can also impact gene expresssion: one protein
that gets phosphorylated by PKA = Creb! Once Creb is
phosphorylated by PKA, it can bind its promoter
element and recruit co-regulators
i.e. CBP which acetylates histones and increases
transcription
CREB also stimulates transcription of Gluconeogenic
enzymes

Metformin
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Figure 34.27 from Marks Basic Medical Biochemistry by Lieberman and Marks
NOTE: TORC2 is different than mTORC2 !!!
Under normal conditions
cAMP (2
ND
messenger) is
cleaved by cAMP
phosphodiesterase to
reduce levels of cAMP
after cell signal activation
Methylxanthines
cAMP phosphodiesterase
inhibitors

Note: ligands tend to be
hydrophilic and cant
diffuse the membrane.
Utilize cell surface
receptors.
Cholera toxin
Gs alpha always on
Pertussis toxin (whooping
cough)
Gi alpha always off

Take home message is an
increase in cAMP in both
cases!!!!
Symptoms differ due to the
tissue specificity of cholera
(GI) and pertussis
(Respiratory)
Cholera Toxin disrupts Gs signaling
Primary target = Gs-alpha; downstream target = adenylyl
cyclase
Chemically modifies Gs so that GTP CANNOT be hydrolyzed to GDP
on the alpha-subunit, so it stays in the ON (GTP-alpha)
configuration
Chemical modification = ADP-ribosylation
Gs stays active increases adenylyl cyclase increases cAMP
constant activation of PKA
Key target for PKA = CFTR channel (Cl- transport
channel) when CFTR active, Cl- leaves intestinal cells
and goes into lumen & H2O follows (osmotic activity)
Elevated PKA high CFTR activity lose lots of H2O causes
DIARRHEA, DEHYDRATION, & DEATH
Pertussis Toxin (Whooping Cough)
Chemically modify Gi so it becomes INACTIVE when
Gi is inactive, there is NO inhibitory mechanism
adenylyl cyclase INCREASES HIGH cAMP levels
Respiratory system
Whooping cough/machine gun-like cough; uncontrollable
coughing fits, vomiting occurs w/ coughing, infant choking
spells
Odorant receptors
Stimulate adenylyl cyclase increase cAMP
cAMP binds Na+ ion channel Na+ causes
depolarization causes changes in membrane potential /
nerve signaling triggers nerve impulse
Role of Transducin (Gt) in Photoreception
The photoreceptor in rod cells of
the retina is a G protein-coupled
receptor called rhodopsin.
Rhodopsin is activated when light
is absorbed by the associated
small molecule retinal.
Rhodopsin then activates the G
protein transducin (Gt); the
subunit stimulates cGMP
phosphodiesterase, leading to
decreased levels of cGMP.
cGMP levels are translated to
nerve impulses by a direct effect of
cGMP on ion channels.
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Gq signaling
Ligand binds receptor
Gq activated
Gq activates PLC-beta
PLC-beta cleaves PIP2 IP3 & DAG
IP3 causes Ca
+2
release from ER
Ca
+2
binds calmodulin alters other pathways
DAG activates PKC (what else does PKC need in order to
be activated?)
PKC stimulates other pathways
REGULATION OF GLYCOGEN METABOLSIM IN THE
LIVER
Regulation of Ca
+2
in electrically excitable
cells
Ryanodine receptors = Ca
+2
-gated-Ca
+2
channels
Open when Ca
+2
binds to them
2 waves of Ca
+2
:
1. Ca
+2
influx from extracellular source via voltage-gated Ca
+2
channels Ca
+2
binds ryanodine receptors on ER
2. ^This triggers Ca
+2
release from the SR into the sarcoplasm (T-
tubules)
Skeletal mm: Ca
+2
binds Troponin-C
Smooth mm: Ca
+2
binds Calmodulin

Malignant Hyperthermia
AD
Sensitivity to anesthetics (i.e. halothane,
succinylcholine)
MUTATION IN RYANODINE RECEPTOR
Life-threatening fever, sustained mm contraction,
hypercatabolism
Increases Ca
+2
in sarcoplasm after drug exposure
dangerously high body temps due to continuous mm
contraction and heat generation
How acetylcholine induces smooth
muscle relaxation
1. Acetylcholine binds to heptahelical receptor and triggers Gq
signaling in the endothelial cells
2. Produce IP3 Calcium is released into the cytoplasm from the sER
3. Calcium binds calmodulin
4. Calmodulin activates Nitric Oxide synthase
5. Nitric Oxide synthase creates NO and citrulline from Arginine
6. NO diffuses out of the endothelial cells and into the smooth muscle
cell
7. NO binds and activates soluble Guanylyl cyclase
8. Guanylyl cyclase synthesizes cGMP from GTP
9. cGMP activates Protein Kinase G (PKG) resulting in smooth
muscle relaxation (Vasodilation)
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NO signalling
NO, erectile dysfunction, and angina
pectoris
Nitroglycerin: for angina (chest pain) releases
constricted coronary arteries
Glycerol trinitrate (Nitroglycerin) decomposes to NO activates
guanylyl cyclase relaxes smooth mm in coronary arteries
Bypasses the NO synthesis process so QUICKER effect
Viagra: inhibits cGMP-phosphodiesterase inhibits
breakdown of cGMP
Helps maintain elevated levels of cGMP elevated levels of active
PKG & increased vasodilation in erectile tissue

The steroid hormone cortisol (hydrocortisone) is the major physiologic
glucocorticoid in humans. Glucocorticoids, such as cortisol, were
named for their ability to raise blood glucose levels. These steroids
are among the counterregulatory hormones that protect the body
from insulin-induced hypoglycemia.
Where is the cellular location of the glucocorticoid receptor?
1. Lumen of the endoplasmic reticulum
2. Lumen of the Golgi complex
3. Inner membrane of the mitochondria
4. In the cytosol
5. On the plasma membrane
Which of the following statements is true about
caffeine?
1. Caffeine directly activates adenylyl cyclase
2. Caffeine inhibits cGMP phosphodiesterase
3. Caffeine inhibits cAMP phosphodiesterase
4. Caffeine inhibits phospholipase C
5. Caffeine activates phosphodiesterase
The phosphorylated form of glycogen
phosphorylase .
1. can be activated in muscle in the
presence of calcium
2. is active
3. is inactive
4. is inactivated by secretin
5. does not exist
How does cholera toxin induce the cell to
secrete salts and fluid into the gut?
1. Chemically modifies adenylyl cyclase
directly so that it can no longer
synthesize cAMP
2. Chemically modifies Gs so that it can
no longer hydrolyze GTP
3. It inhibits Gi
4. Cholera toxin mimics cAMP and binds
to cAMP targets
Epinephrine binding to b-Adrenergic receptors can lead
to increased production of cyclic AMP (cAMP). Which
term best describes cAMP in this situation?
1. Second messenger
2. Endocrines
3. Paracrines
4. Primary messenger
5. Autocrine
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Signaling through enzyme-coupled cell-
surface receptors
Receptors that are kinases or that bind kinases. The kinase
domains are shown in red, and the phosphorylation sites are
indicated with red arrows.
A: Tyrosine kinase receptors.
B: JAK-STAT receptors.
C: Serinethreonine kinase receptors.
Receptor Tyrosine Kinases
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You need to know those marked with a red arrow
Achondroplasia
Mutation in FGFR3 gene
FGFR3 receptor = RTK
Dominant Negative disorder
Most common cause of short-limbed dwarfism small
stature, short-limbs, large head
Failure of long bone growth causes short stature
80% of time, you have a normal couple that gives birth to
a child w/ achondroplasia therefore, 80% of the time,
its a new de novo mutation
Usually, new mutation occurs in sperm more likely to have a
child w/ this mutation when the FATHER is older!

Ras/MAPK Signaling Pathway
1. GRB2 has SH2 domain and binds phosphotyrosine receptor,
localizes it to membrane
2. Sos (a GEF), which is assocd with GRB2, becomes active
3. Sos stimulates Ras (monomeric G-protein) to release GDP
and bind GTP
1. Now Ras is ACTIVE
4. Ras activates RAF (MAPKKK)
5. RAF activates MEK (MAPKK)
6. MEK activates ERK (MAPK)
7. MAPKs (i.e. ERK) phosphorylate TFs (ex. AP-1)
8. TFs induce changes in gene expression and promote a
cell to grow & divide

To shut this off, you need a GAP (GTPase activating
protein hydrolyzes GTP to GDP)

This pathway can be up-regulated in TUMORS
continuous cell proliferation
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Ras - MAPK Signaling Pathway
Noonan Syndrome
Mutation in genes encoding Ras/MAPK signaling
proteins Ras/MAPK defective
i.e. PTPN11, KRAS, SOS, RAF1
AD; short stature, distinct craniofacial features, congenital
CV diseases, developmental delays/intellectual disability,
bleeding tendencies, lymphatic & genitourinary
abnormalities; WEBBING OF THE NECK
This disease has locus heterogeneity various
phenotypes
Noonan Syndrome
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Note: Excessive nuchal skin/webbed
neck is sometimes seen in individuals
with Noonan syndrome
EGF Receptor Tyrosine Kinase-signalling
(Phospholipase C-)
1. Epidermal growth factor (EGF) binds receptor,
extracellular domains dimerize > kinase
domains phosphorylate each other
2. Phospholipase C- binds to activated receptor
protein tyrosine kinases via SH2 domains
3. Tyrosine phosphorylation increases PLC-
activity
4. Stimulates hydrolysis of PIP2 > IP3 and DAG
5. IP3 stimulates Ca2+ release
6. DAG stimulates PKC pathway

Receptor tyrosine kinase (Growth factors)
Nuclear factor of activated T-cells (NFAT)
Activation Pathway
1. Stimulated T cells trigger Phospholipase C- >
Ca
2+
release
2. Ca
2+
binds and activates calmodulin
3. Calmodulin binds and activates calcineurin
4. Calcineurin dephosphorylates NFAT
5. NFAT goes to nucleus and stimulates IL-2 gene
expression
6. Cyclosporin A inhibits calcineurin and is used
as an immunosuppresant (organ transplant) >
NFAT left in phosphorylated state
FIGURE 10.1. from Cell Biology 3
rd
edition by
Bolsover et al
NFAT and Cyclosporin A
RTK activation of PI 3/Kinase/AKT

1. Ligand binds receptor, receptors aggregate and
autophosphorylate each other
2. PI 3 Kinase binds phosphorylated tyrosine on
receptor and becomes active
3. PI 3 Kinase phosphorylates PIP2 to become
PIP3
4. PDK and AKT (PKB) bind PIP3 molecules
5. PDK and mTORC2 then phosporylate and
activate AKT
6. AKT then phosphorylates downstream targets

P-I3 Kinase / Akt Pathway
The PI 3-kinase/Akt pathway
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AKT/FOXO Signaling
Absence of growth factor and Akt
phosphorylation
1. Akt not active
2. FOXO is released from
chaperone 14-3-3 in
cytoplasm
3. Translocates to nucleus
4. Stimulates transcription
factors
5. Functions as trigger for
apoptosis by
upregulating
genes necessary for cell
death
Presence of growth factor and Akt
phosphorylation
1. Akt active
2. Phosphorylates FOXO
3. Creates binding sites for
cytosolic chaperone 14-3-
3
4. Sequeters FOXO in
inactive form in cytoplasm
5. Inhibits FOXO-dependent
transcription
6. No apoptosis
PI 3-Kinase/AKT Signaling and PTEN-
Tumor suppressor
Activated AKT dissociates from plasma
membrane and phosphorylates various targets
PTENs removes phosphate from PIP3 > PIP2>
negative control on AKT activation
Continued activation of PKB/AKT could lead to
increased cell growth and tumor development

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Insulin Receptor
RAS/MAPK PI 3 Kinase
AKT
GLUT 4 to
membrane
GSK off,
Increased
glycogen
synthesis
TSC1/TSC2 off
Increased protein
translation
Rheb on
MTORC 1 on
p70S6K on 4EBF1 off
eIF4E on
FOXO
phosporylated
Decreased
glucose synthesis
PLC-g
Insulin Signaling Pathway
1. Example of Tyrosine Kinase growth factor signaling
2. Insulin receptor is a multi-subunit receptor tyrosine kinase
3. Preformed complex > dimer of two membrane-spanning
pairs
4. Insulin binds receptors > activates receptor
5. Activated insulin receptor binds IRS molecules (insulin
receptor substrates)
6. IRS acts as scaffold and gets phosphorylated at multiple
sites
7. Forms binding sites for proteins with SH2 domains
8. Initiates signal transduction, leading to glucose import,
stimulation of glycogen synthesis, and regulation of gene
expression > activates multiple pathways > 3 key examples:
1. GRB2 in RAS/MAPK pathway
2. phospholipase C
3. PI 3-Kinase

PI 3 kinase/AKT signalling
Activation of AKT (PKB) has four important
consequences;
1. GLUT4 > PM > glucose uptake.
2. AKT phosphorylates glycogen synthase
kinase-3 (GSK-3 deactivated) > increase in
glycogen synthesis
3. Alters TSC/mTOR pathway > increase
protein translation
4. Alters FOXO pathway > decreases glucose
synthesis

Insulin signalling Receptor Tyrosine
Kinase
TSC/mTORC Signaling
1. Receptor binds insulin
2. PI 3 Kinase
3. AKT
4. TSC1/TSC2 off
5. Rheb on
6. MTORC 1 on
7. p70S6K on
8. 4EBF1 off
9. eIF4E on
10. Increased protein translation > cell growth
Tuberous sclerosis
Inherited disorder whose key
features include multiple facial
angiofibromas, hypopigmented
macules, periungual fibromas,
seizures, Shagreen patch, cardiac
rhabdomyoma, and renal lesions.
mutation in either TSC1 or TSC2 >
constitutively off > mTORC1 always
on > uncontrolled cell growth and
proliferation
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Multiple facial angiofibromas
TSC/mTORC Signaling-cell growth
This pathways is sensitive to nutrients and
hormones > cell growth and proliferation
Tumor cells due to up-regulation of this
pathway
Rapamycin; complexes with FKBP12 and
inhibits mTORC1 > inhibits cell proliferation
Immuonosupressant; prevents transplant rejection
TGF receptor signaling (ser/thr kinase
for growth factor signalling)
1. Ligand binds complex of type I/type II receptors
2. Type II receptor P's Type I receptor
3. Smad 2/3 (regulatory)gets P'd > complexes with
Smad4
4. Complex goes into the nucleus
5. Binds to transcriptional factors and modulates
gene expression
1. Pathway off; lower gene expression
2. Pathway on; increases gene expression > grow and
divide (mutation here can lead to colon cancer)
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Some growth factors transduce signals via
receptor serine/threonine kinase
Transforming Growth Factor
Beta receptor is an example of
serine/threonine kinase
TGF ligand binds a complex of
type I and II receptors
Type II receptor phosphorylates
type I receptors
Type I receptors phosphorylates
an R-Smad (Smad2 or Smad3)
R-Smad complexes with Smad4
In the nucleus, the Smad complex
associates with transcription
factors to modulate gene
expression
JAK-STAT signaling (immune system)
1. Cytokine-Receptors already has JAKs bound -
binding of cytokines > receptors cross-link,
activates JAK - JAK will autophosphorylate the
receptor and itself
2. JAKs recruit and phosphorylate STAT1/2
3. STAT1/2 dissociate from receptor and dimerize
via their SH2 domain
4. STAT1/2 dimer translocate to the nucleus, bind
to DNA and other gene regulatory proteins
5. Activate gene transcription

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JAK-STAT Signaling Pathway
Delta-Notch signalling (during
development-cell to cell signalling)
Signals neighboring cells to divide into a different
type of cell
1. Delta binds Notch (receptor)
2. Cytoplasmic tail of Notch is cleaved and acts
as a transcription factor
3. Translocates to nucleus, binds regulatory
factors and gene promotors (DNA)
4. Modulates gene transcription
Wnt/b-catenin signaling (in development)
Absence of Wnt
The Destruction Complex
composed of APC, axin,
GSK3 and CK1
phosphorylates b-catenin
b-catenin is ubiquitylated
and degraded
b-catenin levels kept low in
the cell
Can not modulate gene
expression
Presence of Wnt
Wnt binds and activates
receptors Frizzled and LRP
Dishevelled becomes
activated
The Destruction Complex is
turned off
b-catenin is no longer marked
for destruction and its levels
increase within the nucleus
b-catenin modulates gene
expression
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Wnt/b-catenin signalling
Hedgehog signaling
1. Receptor for Hedgehog is a protein (patched) that
inhibits a second transmembrane protein (Smoothened)
by an unknown mechanism
2. Hedgehog binding inhibits Patched, leads to activation
of Smoothened
3. Initiates a signaling pathway leading to activation of
transcription factor Gli

NF-kB Signaling(immune response)
Latent gene regulatory proteins in cells
Activated during stress, inflammation to protect cells
1. TNF binds the receptor
2. IkB Kinase becomes active and phosphorylates IkB
3. IkB is ubiquitinated and degraded
4. NF-kB is no longer bound to IkB and its nuclear
localization signal is now exposed
5. NF-kB is transported into the nucleus through the
nuclear pores
6. NF-kB binds to promoter elements and modulates
gene expression

NF-B signaling from the TNF receptor
62
Integrin signalling


64
Disruption of growth factor signaling can
lead to cancer (mutations)
Ras mutations are often associated with cancer
Epidermal growth factor (EGF) receptor
mutations can lead to breast cancer,
glioblastoma, and fibrosarcoma (cancer of long
bones)
TGF beta type I receptor mutations occur in 1/3 of
ovarian cancers
TGF beta type II receptor mutations occur in
many colorectal cancers
Smad4 mutation occur ~50% of pancreatic
cancers
Which MAP kinase will the MAP
kinase kinase MEK activate?
1. RAF
2. RAS
3. ERK
4. GRB2
5. SOS
Which G protein below is associated with
the formation of inositol triphosphate?
1. G
q
2. G
s
3. G
i
4. G
t
5. None of the above
Which of the following statements is true
about sildenafil (Viagra)?
1. Viagra primarily inhibits nitric oxide
production
2. Viagra primarily activates guanylyl
cyclase
3. Viagra primarily activates cGMP-
specific phosphodiesterase
4. Viagra primarily activates adenylyl
cyclase
5. Viagra primarily inhibits cGMP-specific
phosphodiesterase
One of the most common proto-oncogenes mutated in
cancer is Ras. Which of the following regulatory proteins
would be directly up-regulated if Ras was constitutively
active in a tumor cell?
1. Raf
2. PI 3-Kinase
3. Adenylyl cyclase
4. SOS
5. Phospholipase Cb
How is Notch activated as a transcriptional
regulator?
1. It is cleaved from the membrane-bound
receptor
2. It is no longer marked for destruction
by APC
3. It is freed from an inhibitor protein
which sequesters Notch in the cytosol
4. It is phosphorylated by a tyrosine
kinase associated with the receptor
5. It binds to Smad 3
A 9-month-old girl is being followed by her pediatrician for dysmorphic
features, hypotonia and delay in reaching motor milestones. Her physician
notes that her head is large. She has frontal bossing and midface
hypoplasia. Her arms and legs are shortened proximally. Although her
parents and three-year-old brother are of normal height, she is short for her
age.
Which receptor does she most likely have mutated?

1. Fibrillin receptor
2. Fibroblast growth factor receptor 3
3. TGF-b receptor 2
4. EGF receptor
5. Endothelial growth factor receptor 3
Which of the following proteins is mutated
in tuberous sclerosis?
1. mTOR
2. Rheb
3. Ras
4. TSC1
5. LKB1
Smad 2 is an R-smad involved with the TGF-b signaling
pathway. Which protein does Smad 2 complex with, after
Smad 2 is phosphorylated? This complex migrates into the
nucleus and alters gene expression.
1. TGF-b receptor I
2. TGF-b receptor II
3. Both TGF-b receptors I & II
4. ERK
5. Smad 4
Which protein is phosphorylated after interferon
stimulates its JAK-associated receptor?
1. Nf-kB
2. STAT
3. b-catenin
4. Notch
5. TGF-b
Which enzyme is responsible for
phosphorylating the phospholipid PIP2
into PIP3?
1. PTEN
2. Ras
3. MAP Kinase
4. Phospholipase Cg
5. PI 3-Kinase
What dephosphorylates
PIP3 back to PIP2?