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Introduction to Signaling Networks
Biophysics 6702, February 2013Jonathan P Butchar
Outline
• General Signaling Concepts• Types of Signaling• Signaling Components
– Receptors and Ligands– Downstream Signaling
• Adaptor and Effector molecules
• Example Signaling Network: FcγR
Outline• General Signaling Concepts
Figure 15-8 Molecular Biology of the Cell (© Garland Science 2008)
Different signals and signal combinations lead to different outcomes
Figure 15-9 Molecular Biology of the Cell (© Garland Science 2008)
A single signal can lead to different outcomes
Figure 15-9 Molecular Biology of the Cell (© Garland Science 2008)
How?
Figure 15-10 Molecular Biology of the Cell (© Garland Science 2008)
Strength of signal can determine outcome
Figure 15-10 Molecular Biology of the Cell (© Garland Science 2008)
How else could a single signal lead to different outcomes?
Figure 15-6 Molecular Biology of the Cell (© Garland Science 2008)
Slow and fast response times
Figure 15-20 Molecular Biology of the Cell (© Garland Science 2008)
Different signals can converge on one target
Figure 15-66 Molecular Biology of the Cell (© Garland Science 2008)
Crosstalk between signaling pathways
Figure 15-51 Molecular Biology of the Cell (© Garland Science 2008)
Cells can adapt to signaling
Figure 15-51 Molecular Biology of the Cell (© Garland Science 2008)
How else might cells adapt?
Figure 15-28a Molecular Biology of the Cell (© Garland Science 2008)
Signals can be amplified
Figure 15-28c Molecular Biology of the Cell (© Garland Science 2008)
or dampened
Figure 15-15 Molecular Biology of the Cell (© Garland Science 2008)
Primary and secondary responses
Figure 15-15 Molecular Biology of the Cell (© Garland Science 2008)
Primary and secondary responsesHow to tell the difference?
Outline• Types of Signaling
Figure 15-4a Molecular Biology of the Cell (© Garland Science 2008)
Figure 15-4b Molecular Biology of the Cell (© Garland Science 2008)
Don’t forget Autocrine signaling
Figure 15-4d Molecular Biology of the Cell (© Garland Science 2008)
Figure 15-4c Molecular Biology of the Cell (© Garland Science 2008)
Figure 15-7 Molecular Biology of the Cell (© Garland Science 2008)
Direct Transmission
Outline• Signaling Components
– Receptors and Ligands
Figure 15-3a Molecular Biology of the Cell (© Garland Science 2008)
Figure 15-3b Molecular Biology of the Cell (© Garland Science 2008)
Numerous types of ligands
• Peptides / Proteins• Steroids• Nucleotides• Fatty Acids• Gases• Mechanical Forces• temperature, etc
How do you get from ligand binding to an intracellular response?
• Ion fluxes• G-protein activation• Enzyme activation (e.g., Phosphorylation)
Figure 15-16a Molecular Biology of the Cell (© Garland Science 2008)
Na+, K+, Ca2+, Cl-
e.g., Cystic Fibrosis is caused by defects in a Chloride channel
There are voltage-gated ion channels too
Figure 15-16b Molecular Biology of the Cell (© Garland Science 2008)
G-proteins: Guanine nucleotide-binding proteins
Figure 15-16c Molecular Biology of the Cell (© Garland Science 2008)
Enzyme-containing or enzyme-linked
Figure 15-53a Molecular Biology of the Cell (© Garland Science 2008)
Example: Receptor Tyrosine Kinases
Figure 15-53b Molecular Biology of the Cell (© Garland Science 2008)
Inhibition of Receptor Tyrosine Kinases
Figure 15-53b Molecular Biology of the Cell (© Garland Science 2008)
Inhibition of Receptor Tyrosine KinasesHow else could you do this?
Figure 15-14b Molecular Biology of the Cell (© Garland Science 2008)
Receptors can be locked in an inactive state
Figure 15-14c Molecular Biology of the Cell (© Garland Science 2008)
…and then unlocked by a ligand
Outline
• Downstream Signaling Components– Signaling, Adaptor and Effector molecules
Figure 15-54 Molecular Biology of the Cell (© Garland Science 2008)
Signaling molecules transduce receptor activation
Figure 15-22 Molecular Biology of the Cell (© Garland Science 2008)
Common domains of signaling molecules
Did I mention this is a really good book?
Proline-rich
What molecules transmit these signals?
• Kinases and Phosphatases• GTP / GDP
Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008)
Kinases and phosphatases
e.g., Receptor Tyrosine Kinase: a receptor AND a kinase
Figure 15-18b Molecular Biology of the Cell (© Garland Science 2008)
Guanosine TriPhosphate and Guanosine DiPhosphate
Remember the G-protein coupled receptor
Figure 15-32 Molecular Biology of the Cell (© Garland Science 2008)
Trimeric G-proteins
• 3 subunits– α, β, γ
Figure 15-19 Molecular Biology of the Cell (© Garland Science 2008)
Monomeric G-proteins
• Most well-known is Ras– small GTPase– downstream Raf binds
only GTP-Ras, which phosphorylates and hence activates Raf
GTPase-activating protein
Guanine nucleotide exchange factor
Figure 15-21c Molecular Biology of the Cell (© Garland Science 2008)
How do all these things get together?
$125.99 at Amazon.com.
Figure 15-21a Molecular Biology of the Cell (© Garland Science 2008)
Scaffolding proteins can aid the interaction of signaling molecules
Figure 15-22 Molecular Biology of the Cell (© Garland Science 2008)
Binding occurs through characteristic domains
• PTB binds phosphotyrosine
• SH2 binds phosphotyrosine
• SH3 binds proline-rich domains
• PH binds phosphoinositides
Proline-rich
Figure 15-21b Molecular Biology of the Cell (© Garland Science 2008)
Signaling molecules can also associate directly with receptors
Figure 15-36 Molecular Biology of the Cell (© Garland Science 2008)
Example: from G-proteins to gene transcription
• Activated receptor• Activated G-protein• Activated Protein Kinase A• The Effector, an activated
transcriptional modulator
Lines are blurry at times
• Some membrane-bound receptors (e.g., glucocorticoid receptors) can go to the nucleus and regulate gene transcription– Both a receptor and an effector
• Phosphorylation can sometimes deactivate rather than activate a protein (e.g., the NF-κB inhibitor IκBα)
Outline• Example Signaling Network: FcγR
Fcγ Receptors bind the Fc portion of IgG
FcγRIIa(CD32a)
FcγRI(CD64)
FcγRIIIa(CD16)
FcγRIIb(CD32b)
γ γ
membrane
ActivatingInhibitory
γ γ
ITIM
ITAM
ITAM
ITAM
ITAM
ITAM
Fcγ Receptors bind the Fc portion of IgG
• Rituximab, Herceptin, etc• Autoantibodies (e.g., rheumatoid arthritis)
http://www.whfreeman.com/immunology/CH01/figure01-04a.gif
PhagocytosisWhat kind of signaling is this?
FcγR activation
• FcγR must be clustered• Phosphorylation drives
downstream events– ImmunoTyrosine-based
Activation Motif
• ITIM phosphorylation dampens FcγR activity
-P
PtdIns 3,4,5P 3
Btk
Ca2+ flux
Phagocytosis
Gene Transcription
Ras/MAPKVav
Akt
ShcGrb2
Sos
Syk PI3-K
-P
FcγR
Immune Complex
Y
Y
YY
YY
Y
NF-κB
Src
Downstream signaling: Ras and PI3K
-P
PtdIns 3,4,5P 3
Btk
Ca2+ flux
Phagocytosis
Gene Transcription
Ras/MAPKVav
Akt
ShcGrb2
SosSyk PI3-K
-P
FcγR
Immune Complex
Y
Y
YY
YY
Y
NF-κB
Src
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
Ras signaling review in under 10 seconds…
• G-protein• small GTPase
2002 edition searchable for free athttp://www.ncbi.nlm.nih.gov/books/NBK21054/
Figure 15-64 Molecular Biology of the Cell (© Garland Science 2008)
Quick PI3K signaling review(phosphoinositide 3-kinase)
Requires membrane localization
There’s crosstalk between Ras and PI3K
-P
PtdIns 3,4,5P 3
Btk
Ca2+ flux
Phagocytosis
Gene Transcription
Ras/MAPKVav
Akt
ShcGrb2
SosSyk PI3-K
-P
FcγR
Immune Complex
Y
Y
YY
YY
Y
NF-κB
Src
srcP
P P
P PIP25 4
SykShc
Grb2Sos
Ras
Erk
PI-3K
PIP35 4
3
Btk
Actin Polymerization
Phagocytosis / ROS / Cytokine
VavRac
Akt
Ca++
PLC
Inflammatory Cytokines (IL-1, TNFα etc)
O2-
How could we modulate FcγR activity?
-P
PtdIns 3,4,5P 3Btk
Ca2+ flux
Phagocytosis
Gene Transcription
Ras /MAPKVav
Akt
Shc
So
s
Syk PI3-K
-P
FcγR
Immune Complex Y
Y
YY
YY
Y
SrcGrb2
Remember, activating and inhibitory receptors
FcγRIIa(CD32a)
FcγRI(CD64)
FcγRIIIa(CD16)
FcγRIIb(CD32b)
γ γ
membrane
ActivatingInhibitory
γ γ
ITIM
ITAM
ITAM
ITAM
ITAM
ITAM
Summary
• Cells and groups of cells possess mechanisms to generate and respond to signals
• Signaling can be autocrine, paracrine, endocrine, synaptic, electrical or mechanical
• Receptors sense numerous types of stimuli and begin cascades that lead to cellular responses
• Observed responses represent an integration of stimuli, both past and present