Introduction to Signaling Networks Biophysics 6702, February 2013 Jonathan 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 Molecular Biology of the Cell (© Garland Science 2008) Strength of signal can determine outcome

Figure 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 Molecular Biology of the Cell (© Garland Science 2008) Different signals can converge on one target

Figure Molecular Biology of the Cell (© Garland Science 2008) Crosstalk between signaling pathways

Figure Molecular Biology of the Cell (© Garland Science 2008) Cells can adapt to signaling

Figure 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 Molecular Biology of the Cell (© Garland Science 2008) Primary and secondary responses

Figure Molecular Biology of the Cell (© Garland Science 2008) Primary and secondary responses How 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 +, Ca 2+, 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 Kinases How 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 Molecular Biology of the Cell (© Garland Science 2008) Signaling molecules transduce receptor activation

Figure 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 Molecular Biology of the Cell (© Garland Science 2008) Trimeric G-proteins 3 subunits –α, β, γ

Figure 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? $ at Amazon.com.

Figure 15-21a Molecular Biology of the Cell (© Garland Science 2008) Scaffolding proteins can aid the interaction of signaling molecules

Figure 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 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 Activating Inhibitory γγ ITIM ITAM

Fcγ Receptors bind the Fc portion of IgG Rituximab, Herceptin, etc Autoantibodies (e.g., rheumatoid arthritis)

Phagocytosis What 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 Ca 2+ flux Phagocytosis Gene Transcription Ras/MAPK Vav Akt Shc Grb2 Sos Syk PI3-K -P FcγR Immune Complex Y Y Y Y Y Y Y NF-κB Src

Downstream signaling: Ras and PI3K -P PtdIns 3,4,5P 3 Btk Ca 2+ flux Phagocytosis Gene Transcription Ras/MAPK Vav Akt Shc Grb2 Sos Syk PI3-K -P FcγR Immune Complex Y Y Y Y Y Y Y NF-κB Src

Figure Molecular Biology of the Cell (© Garland Science 2008) Ras signaling review in under 10 seconds… G-protein small GTPase 2002 edition searchable for free at

Figure 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 Ca 2+ flux Phagocytosis Gene Transcription Ras/MAPK Vav Akt Shc Grb2 Sos Syk PI3-K -P FcγR Immune Complex Y Y Y Y Y Y Y NF-κB Src

src P PP P PIP2 5 4 Syk Shc Grb2 Sos Ras Erk PI-3K PIP Btk Actin Polymerization Phagocytosis / ROS / Cytokine Vav Rac Akt Ca ++ PLC 

Inflammatory Cytokines (IL-1, TNFα etc) O 2- How could we modulate FcγR activity? -P PtdIns 3,4,5P 3 Btk Ca 2+ flux Phagocytosis Gene Transcription Ras/MAPK Vav Akt Shc Sos SykPI3-K -P FcγR Immune Complex Y Y Y Y Y Y Y Src Grb2

Remember, activating and inhibitory receptors FcγRIIa (CD32a) FcγRI (CD64) FcγRIIIa (CD16) FcγRIIb (CD32b) γ γ membrane Activating Inhibitory γγ ITIM 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