1. Cell Signaling Systems 1. General Principles of Information Propagation 2. Signaling Pathways vs Networks Mechanisms and Consequences of Networking: Information Processing

2. Signal Epinephrine Glucagon LH Receptor  -AR GR, LHR Effector Adenylyl Cyclase cAMP ATP Second Messenger Protein Kinase A Phosphorylated Proteins Change in activity of Enzymes, channels, transcription factors Transducer G s heterotrimer cAMP +    A Linear Signaling Pathway Glucose Metabolism Cardiac Contractility Gene Expression

4. Signaling through non-covalent interactions Outside the cell Ligand – receptor interactions Inside the cell 1) Interactions between second messengers (small molecules) and targets cAMP with protein kinase A or cAMP-GEF IP 3 with the IP 3 receptor (ER Ca 2+ channel) 2) Protein-protein interactions GRB and SOS (Ras-GEF) with Ras Ras with Raf

5. Signaling through enzymatic activity GTPases Heterotrimeric G proteins Small GTPases: Ras, Rho/Cdc42, Rap and Rab families Protein Kinases and Phosphatases Ser-Thr Kinases: Protein kinases A and C CaMK-II Tyr-Kinases: EGFR, Src and JAK Phosphatases: PP2A, PP1, Calcineurin, SHP and PTP-1

6. Mechanism of Information Transfer Change in activity state of the upstream component leads to change in activity of the down-stream component. Change is generally vectorial in nature Change can be either activating or inhibiting. This is interaction specific. (e.g. phosphorylation of the target can either increase or decrease its activity)

7. Pathways vs Networks Generally pathways involve simple cascade of reactions leading to information flow. Examples of pathways 1) G protein Pathways 2) RTK–Ras–MAPK pathway 3) Cytokine Receptor –JAK-Stat Pathway

8. The Gq-PLC-  pathway Ram and Iyengar STKE Connections Map

9. Cytokine Receptor JAK-STAT Pathway Aaronson and Horvath STKE Connections Map

10. Johnson STKE Connections Map The Growth Factor Receptor-Ras-MAPK pathway

11. Pathways vs Networks Networks arise from interactions whereby a component of one pathway regulates the activity of a second pathway An example of simple network: Interactions between RTK–Ras–MAPK and RTK-PLC-PKC pathways

12. DAGCa 2+ RTK c-Raf MAPK-1,2MEK-1,2 Grb2 SOS Ras Transcription Factors, Other substrates PLC-  PKC AA cPLA 2 IP 3 GAP A simple signaling network Ability of PKC to regulate Ras/Raf and MAPK to regulate PKC through Phospholipase A2 leads to networking between the two pathways

13. DAGCa 2+ RTK c-Raf MAPK-1,2MEK-1,2 Grb2 SOS Ras Stimulation of Proliferation PLC-  PKC AA cPLA 2 IP 3 Persistent Activation GAP Consequence of Networking: A feedback loop that displays bistability Bhalla and Iyengar(1999) Science 283:381

14. 0 0.05 0.1 0.15 015304560 Activated MAPK (  M) Time (min) Sustained phospho-MAPK levels after brief PDGF stimulus P-MAPK 2 51530406050 Time after wash (min) - Phospho-MAPK 2 (arbitrary units) 0102030405060 Stim Wash Assay Bhalla, Ram and Iyengar (2002) Science297:1018

15. Summary-I 1. Signaling pathways allow for linear flow of information: Such information may processed (i.e. amplified, or dampened). Many important physiological processes are regulated by linear signaling pathways, e.g. Adrenaline regulation of glucose metabolism, visual transduction. 2. Networking arises from the ability of a component of one pathway to interact with and regulate another pathway, or by the same component participating in multiple pathways. Networking results in signal processing so that information is transferred across spatial and temporal domains.

16. General Themes in Heterotrimeric G protein Pathways Most ligands interact with more than one receptor isoform All pathways involve small G proteins Small molecule diffusible messengers are key components in several pathways Importance of Spatial Domains Rockman H.A. et al (2002) Nature 415:206

17. Table 1: Phenotypes of Mice deficient in adrenergic receptor subtypes From Philpp M And Hein L (2004) Pharmacol Ther 101: 65-74 Most ligands interact with more than one receptor isoform

18. Heterotrimeric G protein Pathways Coupling to different receptor isoforms lead to different G protein pathways and different biological effects

19. This abridged table from the human Genome Sequence Paper shows only the subset of relevant proteins Venter et al (2001) Science 291:1304 Such isoform diversity is found for many signaling components. #s of isoforms generally increase with the evolution. Mammals have the most isoforms and the most complex networking

20. Isoforms of cellular components can promote signal integration by having partially overlapping connectivity Jordan, Landau, Iyengar (2000) Cell 103:193

21. Connections from heterotrimeric to small GTPases increase the number of pathways G proteins can regulate and thus increase the effects GPCRs have on cellular function

22. Jordan, Landau, Iyengar (2000) Cell 103:193 The same small GTPase regulates multiple functions through different effectors

23. Signal Epinephrine Glucagon LH Receptor  -AR GR, LHR Effector Adenylyl Cyclase cAMP ATP Second Messenger Protein Kinase A Phosphorylated Proteins Change in activity of Enzymes, channels, transcription factors Transducer G s heterotrimer cAMP +    A Linear Signaling Pathway Glucose Metabolism Cardiac Contractility Gene Expression

24. Small Molecule Messengers and Spatial Domains Signal Receptor Adenylyl Cyclase cAMP ATP GsGs    cAMP is a diffusible second messenger ( ~600 Da) Does it diffuse through the cell or are there selected regions in the cell with high concentrations of cAMP?

25. A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell Thomas C. Rich*, Kent A. Fagan, Tonia E. Tse*, Jerome Schaack, Dermot M. F. Cooper, and Jeffrey W. Karpen*, ( 2001) PNAS 98:13049 Study compares local levels of cAMP by using the cyclic nucleotide gated channel as the a cAMP biosensor and compares cAMP levels near the plasma membrane by measurement with the biosensor to cAMP levels in the whole cell by biochemical (radioisotope) labeling measurements

26. Copyright ©2001 by the National Academy of Sciences Rich, Thomas C. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 13049-13054 Fig. 1. Distinct cAMP signals measured in different subcellular compartments

27. Copyright ©2001 by the National Academy of Sciences Rich, Thomas C. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 13049-13054 Fig. 4. A quantitative description of the localized transient cAMP response and the total cellular cAMP accumulation

28. Discrete Microdomains with High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes Manuela Zaccolo* and Tullio Pozzan (2002) Science 295:1711 Live Cell Imaging of cAMP using Using Fluorescence Resonance Energy Transfer (FRET)

29. Figure 2 from Manuela Zaccolo and Tullio Pozzan (2002) Science 295:1711 cAMP microdomains visualized by FRET in rat cardiac myocytes

30. Calcium Microdomains in Aspiny Dendrites Jesse H. Goldberg Gabor Tamas Dmitriy Aronov and Rafael Yuste (2003) Neuron 40: 807-821 Imaging of Ca 2+ in the dendrites of interneurons in tissues slices using fluorescent probes (Fluo-4) by two-photon microscopy The neurons are spontaneously activated or electrically stimulated and imaged using fluorescent probes for Ca 2+.

31. The Ca 2+ microdomains are localized and dynamic Fig 2 Jesse H. Goldberg Gabor Tamas Dmitriy Aronov and Rafael Yuste (2003) Neuron 40: 807-821

32. The Ca 2+ microdomains in the dendrites of the interneurons used in the Yuste study are due to: 1) Calcium inflow through the calcium permeable AMPA channels 2)Extrusion of Ca 2+ by the Na + /Ca 2+ exchanger

33. Summary II 1.Through different receptor isoforms, ligands for GPCRs couple to different signaling pathways. These pathways lead to different physiological effects. 2. The coupling between the heterotrimeric G proteins and small GTPases can lead to signal routing to multiple effectors and thus evoke several responses. 3. Diffusible small molecules are often constrained to spatial domains and thus allow the stimulus to achieve specificity of physiological effects.