Introduction to Signaling Networks

Biophysics 6702, February 2013Jonathan P Butchar

jon.butchar@osumc.edu

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