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Signal Transduction
1
Reference:
Molecular Biology of the cell 4th or 5th ed.
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Why is signaling important?
 Allows cells to respond to external stimuli such
as:
a)
b)
c)
d)
e)
f)
g)
h)
i)
j)
Cytokines
Growth Factors
Hormones
Tissue Repair or Remodeling
Other cells
Stress
Tissue Specific Regulation
Regulate Differentiation and Development
Immune Response
Pathogens
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Signaling is a process of cellular
communication
 Signals from outside to the inside result in changes to
the cell:
1. Gene induction/suppression
2. Differentiation/Development
3. Protein secretion
4. Surface marker changes
5. Changes in cellular distribution
6. Environmental changes
7. Apoptosis
8. Proliferation
9. Motility
10.Destruction of foreign invaders
11.Destruction of aberrant cells
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External signaling can be:
1.
2.
Molecules involved in cell-cell and cell-matrix
interactions.
Autocrine signaling: signaling molecules released by a
cell and only affect itself (e.g. many growth factors).
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External signaling can be:
3.
4.
Paracrine signaling: signaling molecules released by a
cell only affect target cells in close proximity to it
(e.g. neurotransmitter and neurohormones).
Endocrine signaling: signaling molecules (hormones)
synthesized by cells of the endocrine organs - act on
target cells distant from their site of synthesis.
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General model for signaling
Receptor – Ligand mediated
signaling from outside to
inside
Cascade of events (2nd
Messengers --involving a
number of different enzymes
[Ca2+, kinases, phosphatases,
adapter proteins, etc.)
Cellular Changes
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Transmembrane Receptors
 Cell-surface receptors can be
categorized into four classes:
1. Receptor with intinsic
enzymatic activity
 Tyrosine kinase receptors (EGF,
insulin, PDGF)
2. G-protein-coupled receptor
 e.g. receptors for epinephrine, serotonin and glucagon
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Transmembrane Receptors
3. Receptor with an
associated enzyme
 e.g. cytokine receptors,
receptors for growth
hormone and interferons
 Receptor guanylyl cyclases
(atrial natriuretic peptide)
4. Ion-channel-linked
receptor
 e.g. neurotransmittergated ion channels
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TRANSDUCTION
 Intracellular signaling pathways typically involve
phosphorylation cascades that are reversibly and tightly
controlled by protein kinases and protein phosphatases.
 Kinases and phosphatases can be divided into:
1. transmembrane proteins or intracellular proteins.
2. serine/threonine-specific or tyrosine-specific (but also a class of
dual-specific)
 Tyrosine phosphorylation is rare in the cell
 only <0.1% of total protein phosphorylation
 But important in cellular regulation.
 Their importance is evident from the fact that many protein
tyrosine kinases (PTK) are encoded by proto-oncogenes
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Receptor protein tyrosine kinase
(RTKs)
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Receptor protein tyrosine kinase (RTKs)
Extracellular region
Typically several hundred aa)
Ligand binds to the extracellular domain
 Most known ligands are secreted soluble proteins
 Membrane-bound and extracellular matrix-bound
ligands can also activate receptor
Transmembrane region
All have a single hydrophobic transmembrane
region followed by a few basic amino acids.)
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Receptor protein tyrosine kinase (RTKs)
 Cytoplasmic region
1. Contains a protein kinase catalytic domain (PTK),
 conserved in sequence of ~250 aa in length
(conservation from 32-95%).
 Contains a major tyrosine phosphorylation site
(its phosphorylation is required for kinase
activation in many cases)
2. A C-terminal region.
 varies from a few up to 200 aa in length
 most of the tyrosine phosphorylation occurs here.
 Protein kinase activity is stimulated by binding of
ligands to the extracellular side
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Receptor protein tyrosine kinase-initiated
signal transduction
Ligand binding
Receptor oligomerization
tyrosine autophosphorylation of the receptor
subunits
P
P
P
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Receptor protein tyrosine kinase-initiated
signal transduction
 Autophosphorylation of receptors serves two purposes:
1. activates catalytic activity of the PTK.
P
P
Kinase activation
P
P
Binds other proteins
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Signaling
2. changes the conformation of the receptor that allows it to bind to
next cytoplasmic signalling molecules in the cascade.
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(I) Ligand binding induces receptor
oligomerization
1.
2.
3.
4.
Ligand is a dimer
Ligands cluster on scaffolding
Ligands cluster in signaling cell
Ligands induce receptor-receptor interaction
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Examples: PDGF (platelet-derived growth
factor)
PDGF are dimeric - homodimers or heterodimers of
A and B chains.
 PDGF A chain binds only a PDGF receptor
 B chain binds both a and b receptors
 Different composition of the PDGF appears to have different
cellular responses.
A A
A B
A B
B B
B B
B B
a a
a a
a b
a a
a b
b b
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 Apart from the ligands, the extracellular
domains of the receptors are also involved
in the dimer formation
Examples: EGF (epidermal growth factor)
 Ligands are monomeric.
 Ligands induce both homo- and heterodimers of their
receptors.
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(II) Tyrosine phosphorylation of receptors
 Ligand binding  Receptor oligomerization 
Juxtapositioning of the cytoplasmic domains of the
receptors  Conformational changes
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The conformational changes allow Mg2+-ATP to
bind the major autophosphorylation site in PTK
(normally buried in the active site)
initial trans-phosphorylation occurs on a tyrosine residue
in the other monomer of the receptor complex (Tyr857
in PDGF receptor)
P
P
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Other Tyr residues in the receptors can now be
phosphorylated by the activated receptor PTK
These phosphorylations serve as molecular
switches to specifically bind cytoplasmic signaling
molecules
P
P
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P
P
P
P
P
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(III) Interaction of receptors with
cytoplasmic proteins
The next step in PTK-mediated
signaling involves interaction
with cytoplasmic proteins that
contain protein-protein
interaction modules.
CONCEPT
Protein modules direct specific
interactions in signal transduction
pathways.
Various modules are frequently
found in the same proteins
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P
P
P
P
Grb2
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(III) Interaction of receptors with
cytoplasmic proteins
RTKs signal via their
phosphorylated
tyrosine residues
The phosphotyrosines
form binding sites to
which subsequent
signaling and
scaffolding proteins
bind to propagate the
signal
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Signal Transduction by the SRC
Family
SRC family of Protein tyrosine kinases:
8 known members of the family:
(SRC, LCK, BLK, HCK, FGR, YES, LYN, FYN )
60-75% amino acid identity between them (outside
the unique region)
Sequences:
myristylation sequence
unique region
SH3 domain
SH2 domain
catalytic domain
regulatory region
Y
Unique
SH3
SH2
Y
Protein kinase
Membrane
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SH2 (SRC Homology 2) domain
SH2 binds to phosphotyrosine and the immediate
C-terminal residues (3-5) in a sequence-specific
fashion
the autophosphorylated tyrosine residue in a receptor
PTK binds specifically to one or more SH2-containing
proteins, but may not bind to other SH2-containing
proteins
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SH2 (SRC Homology 2) domain
One side of the pocket is lined with
conserved basic aa and binds the
phosphotyrosine
The other side of the pocket is
more variable and allows specific
recognition of the residues at the
C-terminal of the phosphotyrosine.
Variations in the nature of the
hydrophobic socket in different
SH2 domains allow them to bind
to phosphotyrosine adjacent to
different sequences
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Functions of SRC family
Potential substrates of SRC
1. Signal transducing proteins
 many potential substrates are identified
2. Cytoskeletal proteins
on activation, a portion of SRC become associated
with cytoskeleton.
nonactivated SRC and nontransforming mutants of vSRC are not associated with cytoskeleton.
transformation is associated with large changes in
cytoskeleton organization
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Turning off or quenching of receptor PTK
signaling
1. Dephosphorylation
 Tyrosine phosphatases
reverse the effects of
Tyr phosphorylation
 They are both soluble
and receptor-like
 Some are constitutive,
most are regulated
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Turning off or quenching of receptor PTK
signaling
2. Receptor
internalization
 endocytosis, may be
autophosphorylationmediated
3. Negative feedback
loop by
phosphorylation
P
P
P
P
P
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P
P
P
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