SIGNAL

TRANSDUCTION
David Henkin

Cell signalling the general

Glucocorticoid / Thyroid hormone

signalling

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-membranebound 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

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

Regulation of glycogen metabolism by Gs

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

10

Metformin

Figure 34.27 from Marks Basic Medical Biochemistry by Lieberman and Marks
NOTE: TORC2 is different than mTORC2 !!!

Under normal conditions

cAMP (2ND 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

15

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.

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 (Ttubules)

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.
2.
3.
4.
5.
6.
7.
8.
9.

20

Acetylcholine binds to heptahelical receptor and triggers Gq

signaling in the endothelial cells
Produce IP3 Calcium is released into the cytoplasm from the sER
Calcium binds calmodulin
Calmodulin activates Nitric Oxide synthase
Nitric Oxide synthase creates NO and citrulline from Arginine
NO diffuses out of the endothelial cells and into the smooth muscle
cell
NO binds and activates soluble Guanylyl cyclase
Guanylyl cyclase synthesizes cGMP from GTP
cGMP activates Protein Kinase G (PKG) resulting in smooth
muscle relaxation (Vasodilation)

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.
2.
3.
4.
5.

can be activated in muscle in the

presence of calcium
is active
is inactive
is inactivated by secretin
does not exist

How does cholera toxin induce the cell to

secrete salts and fluid into the gut?
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
1.

Epinephrine binding to b-Adrenergic receptors can lead

to increased production of cyclic AMP (cAMP). Which
term best describes cAMP in this situation?
1.
2.
3.
4.
5.

Second messenger
Endocrines
Paracrines
Primary messenger
Autocrine

28

Signaling through enzyme-coupled cellsurface 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.

29

Receptor Tyrosine Kinases

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.
2.
3.

GRB2 has SH2 domain and binds phosphotyrosine receptor,

localizes it to membrane
Sos (a GEF), which is assocd with GRB2, becomes active
Sos stimulates Ras (monomeric G-protein) to release GDP
and bind GTP
1.

4.
5.
6.
7.
8.

Now Ras is ACTIVE

Ras activates RAF (MAPKKK)

RAF activates MEK (MAPKK)
MEK activates ERK (MAPK)
MAPKs (i.e. ERK) phosphorylate TFs (ex. AP-1)
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

32

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

34

Noonan Syndrome

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,

2.
3.

4.
5.
6.

extracellular domains dimerize > kinase

domains phosphorylate each other
Phospholipase C- binds to activated receptor
protein tyrosine kinases via SH2 domains
Tyrosine phosphorylation increases PLC-
activity
Stimulates hydrolysis of PIP2 > IP3 and DAG
IP3 stimulates Ca2+ release
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- >
2.
3.

4.
5.

6.

Ca2+ release
Ca2+ binds and activates calmodulin
Calmodulin binds and activates calcineurin
Calcineurin dephosphorylates NFAT
NFAT goes to nucleus and stimulates IL-2 gene
expression
Cyclosporin A inhibits calcineurin and is used
as an immunosuppresant (organ transplant) >
NFAT left in phosphorylated state

NFAT and Cyclosporin A

FIGURE 10.1. from Cell Biology 3rd edition by

Bolsover et al

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

41

The PI 3-kinase/Akt pathway

AKT/FOXO Signaling
Absence of growth factor and Akt
phosphorylation

Akt not active

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
1.
2.

Presence of growth factor and Akt

phosphorylation
1.
2.
3.

4.

5.
6.

Akt active
Phosphorylates FOXO
Creates binding sites for
cytosolic chaperone 14-33
Sequeters FOXO in
inactive form in cytoplasm
Inhibits FOXO-dependent
transcription
No apoptosis

PI 3-Kinase/AKT Signaling and PTENTumor 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

44
Insulin Receptor

RAS/MAPK

PI 3 Kinase

PLC-g

AKT

GLUT 4 to
membrane

GSK off,
Increased
glycogen
synthesis

TSC1/TSC2 off
Increased protein
translation

FOXO
phosporylated
Decreased
glucose synthesis

Rheb on

MTORC 1 on

p70S6K on

4EBF1 off

eIF4E on

Insulin Signaling Pathway

Example of Tyrosine Kinase growth factor signaling
Insulin receptor is a multi-subunit receptor tyrosine kinase
Preformed complex > dimer of two membrane-spanning
pairs
Insulin binds receptors > activates receptor
Activated insulin receptor binds IRS molecules (insulin
receptor substrates)
IRS acts as scaffold and gets phosphorylated at multiple
sites
Forms binding sites for proteins with SH2 domains
Initiates signal transduction, leading to glucose import,
stimulation of glycogen synthesis, and regulation of gene
expression > activates multiple pathways > 3 key examples:

1.
2.
3.
4.
5.

6.
7.
8.

1.
2.
3.

GRB2 in RAS/MAPK pathway

phospholipase C
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

50

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

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)

53

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 -

2.
3.

4.
5.

binding of cytokines > receptors cross-link,

activates JAK - JAK will autophosphorylate the
receptor and itself
JAKs recruit and phosphorylate STAT1/2
STAT1/2 dissociate from receptor and dimerize
via their SH2 domain
STAT1/2 dimer translocate to the nucleus, bind
to DNA and other gene regulatory proteins
Activate gene transcription

JAK-STAT Signaling Pathway

55

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

58

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

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

62

NF-B signaling from the TNF receptor

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.
2.
3.
4.

5.

RAF
RAS
ERK
GRB2
SOS

Which G protein below is associated with

the formation of inositol triphosphate?
1.
2.
3.
4.
5.

Gq
Gs
Gi
Gt
None of the above

Which of the following statements is true

about sildenafil (Viagra)?
1. Viagra primarily inhibits nitric oxide

2.
3.
4.
5.

production
Viagra primarily activates guanylyl
cyclase
Viagra primarily activates cGMPspecific phosphodiesterase
Viagra primarily activates adenylyl
cyclase
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.
2.

3.
4.
5.

Raf
PI 3-Kinase
Adenylyl cyclase
SOS
Phospholipase Cb

How is Notch activated as a transcriptional

regulator?
1. It is cleaved from the membrane-bound
2.
3.
4.

5.

receptor
It is no longer marked for destruction
by APC
It is freed from an inhibitor protein
which sequesters Notch in the cytosol
It is phosphorylated by a tyrosine
kinase associated with the receptor
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.

2.
3.
4.
5.

Fibrillin receptor
Fibroblast growth factor receptor 3
TGF-b receptor 2
EGF receptor
Endothelial growth factor receptor 3

Which of the following proteins is mutated

in tuberous sclerosis?
1.
2.
3.
4.
5.

mTOR
Rheb
Ras
TSC1
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.
2.
3.
4.
5.

TGF-b receptor I
TGF-b receptor II
Both TGF-b receptors I & II
ERK
Smad 4

Which protein is phosphorylated after interferon

stimulates its JAK-associated receptor?
Nf-kB
2. STAT
3. b-catenin
4. Notch
5. TGF-b
1.

Which enzyme is responsible for

phosphorylating the phospholipid PIP2
into PIP3?
1.
2.
3.
4.
5.

PTEN
Ras
MAP Kinase
Phospholipase Cg
PI 3-Kinase

What dephosphorylates
PIP3 back to PIP2?