1. Introduction to Signaling Networks Biophysics 6702, February 2013 Jonathan P Butchar jon.butchar@osumc.edu

2. Outline General Signaling Concepts Types of Signaling Signaling Components –Receptors and Ligands –Downstream Signaling Adaptor and Effector molecules Example Signaling Network: FcγR

3. Outline General Signaling Concepts

4. Figure 15-8 Molecular Biology of the Cell (© Garland Science 2008) Different signals and signal combinations lead to different outcomes

5. Figure 15-9 Molecular Biology of the Cell (© Garland Science 2008) A single signal can lead to different outcomes

6. Figure 15-9 Molecular Biology of the Cell (© Garland Science 2008) How?

7. Figure 15-10 Molecular Biology of the Cell (© Garland Science 2008) Strength of signal can determine outcome

8. Figure 15-10 Molecular Biology of the Cell (© Garland Science 2008) How else could a single signal lead to different outcomes?

9. Figure 15-6 Molecular Biology of the Cell (© Garland Science 2008) Slow and fast response times

10. Figure 15-20 Molecular Biology of the Cell (© Garland Science 2008) Different signals can converge on one target

11. Figure 15-66 Molecular Biology of the Cell (© Garland Science 2008) Crosstalk between signaling pathways

12. Figure 15-51 Molecular Biology of the Cell (© Garland Science 2008) Cells can adapt to signaling

13. Figure 15-51 Molecular Biology of the Cell (© Garland Science 2008) How else might cells adapt?

14. Figure 15-28a Molecular Biology of the Cell (© Garland Science 2008) Signals can be amplified

15. Figure 15-28c Molecular Biology of the Cell (© Garland Science 2008) or dampened

16. Figure 15-15 Molecular Biology of the Cell (© Garland Science 2008) Primary and secondary responses

17. Figure 15-15 Molecular Biology of the Cell (© Garland Science 2008) Primary and secondary responses How to tell the difference?

18. Outline Types of Signaling

19. Figure 15-4a Molecular Biology of the Cell (© Garland Science 2008)

20. Figure 15-4b Molecular Biology of the Cell (© Garland Science 2008) Don’t forget Autocrine signaling

21. Figure 15-4d Molecular Biology of the Cell (© Garland Science 2008)

22. Figure 15-4c Molecular Biology of the Cell (© Garland Science 2008)

23. Figure 15-7 Molecular Biology of the Cell (© Garland Science 2008) Direct Transmission

24. Outline Signaling Components –Receptors and Ligands

25. Figure 15-3a Molecular Biology of the Cell (© Garland Science 2008)

26. Figure 15-3b Molecular Biology of the Cell (© Garland Science 2008)

27. Numerous types of ligands Peptides / Proteins Steroids Nucleotides Fatty Acids Gases Mechanical Forces temperature, etc

28. How do you get from ligand binding to an intracellular response? Ion fluxes G-protein activation Enzyme activation (e.g., Phosphorylation)

29. 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

30. Figure 15-16b Molecular Biology of the Cell (© Garland Science 2008) G-proteins: Guanine nucleotide-binding proteins

31. Figure 15-16c Molecular Biology of the Cell (© Garland Science 2008) Enzyme-containing or enzyme-linked

32. Figure 15-53a Molecular Biology of the Cell (© Garland Science 2008) Example: Receptor Tyrosine Kinases

33. Figure 15-53b Molecular Biology of the Cell (© Garland Science 2008) Inhibition of Receptor Tyrosine Kinases

34. Figure 15-53b Molecular Biology of the Cell (© Garland Science 2008) Inhibition of Receptor Tyrosine Kinases How else could you do this?

35. Figure 15-14b Molecular Biology of the Cell (© Garland Science 2008) Receptors can be locked in an inactive state

36. Figure 15-14c Molecular Biology of the Cell (© Garland Science 2008) …and then unlocked by a ligand

37. Outline Downstream Signaling Components –Signaling, Adaptor and Effector molecules

38. Figure 15-54 Molecular Biology of the Cell (© Garland Science 2008) Signaling molecules transduce receptor activation

39. 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

40. What molecules transmit these signals? Kinases and Phosphatases GTP / GDP

41. Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008) Kinases and phosphatases e.g., Receptor Tyrosine Kinase: a receptor AND a kinase

42. Figure 15-18b Molecular Biology of the Cell (© Garland Science 2008) Guanosine TriPhosphate and Guanosine DiPhosphate Remember the G-protein coupled receptor

43. Figure 15-32 Molecular Biology of the Cell (© Garland Science 2008) Trimeric G-proteins 3 subunits –α, β, γ

44. 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

45. Figure 15-21c Molecular Biology of the Cell (© Garland Science 2008) How do all these things get together? $125.99 at Amazon.com.

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

47. 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

48. Figure 15-21b Molecular Biology of the Cell (© Garland Science 2008) Signaling molecules can also associate directly with receptors

49. 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

50. 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)

51. Outline Example Signaling Network: FcγR

52. Fcγ Receptors bind the Fc portion of IgG FcγRIIa (CD32a) FcγRI (CD64) FcγRIIIa (CD16) FcγRIIb (CD32b) γ γ membrane Activating Inhibitory γγ ITIM ITAM

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

54. http://www.whfreeman.com/immunology/CH01/figure01-04a.gif Phagocytosis What kind of signaling is this?

55. 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

56. 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

57. 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 at http://www.ncbi.nlm.nih.gov/books/NBK21054/

58. Figure 15-64 Molecular Biology of the Cell (© Garland Science 2008) Quick PI3K signaling review (phosphoinositide 3-kinase) Requires membrane localization

59. 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

60. src P PP P PIP2 5 4 Syk Shc Grb2 Sos Ras Erk PI-3K PIP3 5 4 3 Btk Actin Polymerization Phagocytosis / ROS / Cytokine Vav Rac Akt Ca ++ PLC 

61. 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

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

63. 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