1. Signal Transduction I Receptors & Ligands

2. Signal Transduction

3. Signal Transduction Pathway Overview agonists receptors 2 nd messengers cytoplasmic transducers/ effectors targets

4. The Usual Suspects

5. Seven Lively Helices

6. Seven-Helix Receptor Family All 7-pass receptors interact with heterotrimeric G-proteins (  ) ligand binding triggers release of regulatory  subunits & GDP  GTP exchange  G-protein subunit then activates adenyl cyclase & phopholipases to produce 2 nd messengers cAMP & DAG respectively These 2 nd messengers activate PKA & PKC respectively which phosphorylate targets & the C-term of 7-pass receptor

7. Receptor Tyrosine Kinases

8. Fig. 24-4 Copyright 2008 by Saunders/Elsevier. All rights reserved.

9. Receptor Tyrosine Kinase Families

10. Master of My Domains

11. RTK Signaling ligand binding induces dimerization Transphosphorylation phosphorylated residues serve as binding motifs for transducer proteins

12. Activation of Receptor Tyrosine Kinases by Dimerization Fig. 24-5 Copyright 2008 by Saunders/Elsevier. All rights reserved.

13. RTK- Ras Pathway GAP Grb2,Shc GEF like Sos GEF

14. Jak Activation

15. Receptor-JAK-Stat pathway

16. Activation of Receptor Serine/Threonine Kinases Fig. 24-8 Copyright 2008 by Saunders/Elsevier. All rights reserved.

17. Receptor S/T Kinases Fig. 24-8 Copyright 2008 by Saunders/Elsevier. All rights reserved.

18. Receptor Guanylyl Cyclases binding of ligand induces guanylyl cyclase activity These can be TM or cytoplasmic receptors atrial natriuretic factor receptor is TM NO receptor is cytoplasmic

19. TNF Receptor Family All transduce survival/death signals

20. Sphinogomyelinase/ ceramide signaling pathways Activator: TNF Receptor: TNFR Transducer: Sphingomyelinase Second messenger: Ceramide Targets: PKC, PP2A, protein kinase Fig. 26-11 Copyright 2008 by Saunders/Elsevier. All rights reserved.

21. Toll-like Receptors Fig. 24-11 Copyright 2008 by Saunders/Elsevier. All rights reserved.

22. Wnt-  -catenin pathway

23. Hedgehog Pathway

24. Juxtacrine Signaling: Notch Pathway

25. Ephrin-Eph Receptor Signaling

26. Secreted Signaling Molecules FGF Family Hedgehog Family Wnt Family TGF  Superfamily –TGF  Family –Activin Family –BMP Family EGF Family PDGF Family Retinoids HGF/Scatter Factor Neurotrophins Semaphorins Cytokines

27. Receptors for Soluble Signaling Molecules Receptor Tyrosine Kinase (RTK) pathway –FGFR, EGFR, PDGFR –Receptor coupled to ras signal transduction cascade Smad pathway –Bipartite Serine/Threonine Kinase Receptors –AcRI/II, TGF  RI/II, BMPRI/IIs –Receptors activate Smad transcription factors Wnt-  -catenin pathway –Frizzled family of receptors –Activation of pathway allows  -catenin to enter nucleus JAK-STAT pathway –Peptide hormone or cytokine receptor coupled to a Jak cytoplasmic tyrosine kinase –Jaks activate STAT transcription factors Hedgehog Pathway –Patched and smoothened co- receptors –Activation of pathway converts Ci txn’l repressor to activator Steroids/Retinoids –Nuclear/DNA binding receptors –Hormone permits entry into nucleus, alters DNA binding conformation, or allows interaction with co-txn factors

28. Cell Associated Signaling Molecules ECM (ligand) –Fibronectin –Laminin –Type IV collagen Integrins (receptor) CAMs (receptor) Notch Family (receptor) Delta Family (ligand) Eph Family (receptor) Ephrin Family (ligand)

29. Generalized Signal Transduction

30. Signal Transduction II Transduction Proteins & Signal Transduction by Phosphorylation

31. Major Signaling Highways Growth Factor/Cytokine/Adhesion –RTK –RS/TK –JAK –FAKs Steroid Hormone –Intracellular DNA binding proteins Virtually everything else –7-pass TM receptors

32. Cell Signalling via Membrane Receptors Signal Transduction Pathways Protein-Protein Interactions Regulated by phosphorylation

33. It's All About Phosphorylation RTKs and RS/TK directly phosphorylate each other and substrates upon ligand binding Ligand activates linked TKs indirectly 7TM receptors activate non-receptor S/T kinases

34. Typical Iconic Domain Representations and Family Groupings Receptor Non- Receptor

35. Kinase Family Tree

36. Effects of phosphorylation on protein structure & function 1. Steric interference with substrate or ligand binding isocitrate dehydrogenase: blocks substrate binding ADF/cofilin: blocks actin binding 2. Change in conformation activates many kinases and glycogen phosphorylase 3. Creation of a binding site tyrosine phosphorylation: sites for SH3 and PTB domains serine phosphorylation: site for 14-3-3 domains 4. Targeting phosphorylation of cytoplasmic transcription factors targets Smads and STATs to the nucleus Copyright 2008 by Saunders/Elsevier. All rights reserved.

37. Steric blocking: isocitrate dehydrogenase Unphosphorylated, active Phosphorylated, inactive Isocitrate Fig. 25-2 Copyright 2008 by Saunders/Elsevier. All rights reserved.

38. Insulin receptor tyrosine kinase Receptor tyrosine kinase Fig. 25-3 Copyright 2008 by Saunders/Elsevier. All rights reserved.

39. Src activation http://www.garlandscience.com/garlandscie nce_resources/resource_detail.jsf?landing =student&resource_id=9780815340782_C H06_QTM01

40. What goes on must come off Phosphatase Families Dual

41. Signal Transduction Downstream of Receptor Kinases & Receptor-Linked Kinases Adaptors, Small GTPases, MAPKs

42. Signal Transduction Paths Downstream of Tyrosine Kinases

43. Ras-RTK Signaling or MAP Pathway Fig 29-6

44. RTK- Ras - MAPK Pathway GAP Grb2,Shc GEF like Sos GEF

45. Insulin Triggers Uptake of Glucose – How? Outputs: (1) multiple pathways to the nucleus (2) translocation of GLUT4 to the plasma membrane

46. RTK Signal Transduction Components The Receptor Adaptors, GAPs, GEFs, –All contain SH2 domains that bind P-Y (also have SH3) –Adaptors Shc, Grb2, Nck, Crk –GAPs Ras-GAP, p120 GAP –GEFs Vav, SOS, RasGRF, Dbl –Recruit/activate p21 GTPase family members – (Ras family) Ras, Rho, Cdc42, Rac PI3K, PLC , –Generate 2 nd messengers PLC  - diacylglycerol (DAG)+ inositol phosphate (IP 2, IP 3 ) PI3K – phosphoinositols (PIP 2, PIP 3 )

47. Src – The Prototypical Non-Receptor Tyrosine Kinase SH1 – tyrosine kinase domain SH2 – phosphotyrosine binding domain SH3 – polyproline binding domain SH2 and 3 domains have been found in many other signal transduction proteins and all have a conserved structure and functionality SH1 domain found in all tyrosine kinases

48. Src family of non-receptor tyrosine kinases (Lck in T-lymphocytes) Kinase domain SH3 binds poly-proline helix SH2 binds a phosphotyrosine Phosphorylation inhibits activity Phosphorylation stimulates activity Copyright 2008 by Saunders/Elsevier. All rights reserved.

49. Signaling proteins with adapter domains Copyright 2008 by Saunders/Elsevier. All rights reserved.

50. Adaptors: Masters of their Domains phosphotyrosine binding domains polyproline binding domains

51. Domains that bind phosphorylated ligands Phosphotyrosine peptides PhosphoinosidtidesPhosphoserine peptides Head group Fig. 25-11 Copyright 2008 by Saunders/Elsevier. All rights reserved.

52. Ligands for adapter protein domains Copyright 2008 by Saunders/Elsevier. All rights reserved.

53. Domains that bind other ligands Fig. 25-11 Copyright 2008 by Saunders/Elsevier. All rights reserved.

54. Ligands for adapter protein domains Copyright 2008 by Saunders/Elsevier. All rights reserved.

55. Monomeric GTPase Super Family

56. Small Monomeric GTPases Dramatically different conformations of proteins depending on ligand –GTP vs GDP All have MW ~21KDa –p21 Ras Family –Ras, Rho, Cdc42 Rab family –Rab, Arf, Sar See Table 27-3 for others Fig 27-7

57. Activation Cycle of GTPase Fig27-8

58. GAPs & GEFs Have adaptor domains (SH2, SH3, PH, PTB) GEF – stimulate release of GDP from GTPase GAP – stimulate hydrolysis of GTP by GTPase Fig 27-12

59. Activated Ras Triggers MAP Kinase Cascade MAPK = Mitogen Activated Protein Kinases –ERK1, ERK2, etc = Extracellular signal Regulated Kinase MAPK is activated by a kinase called variably MAPK Kinase (MAPKK) or MEK (MAPK/ERK Kinase) MEK is activated by a kinase called Raf (Ras activated factor) (or MAPK Kinase Kinase) Raf is activated by Ras

60. HD – histone fold domain DH – Dbl homology, Difuse B-cell Lymphoma PH – Plekstrin homology REM - Ras Exchange motif CDC – cell division cycle or cyclin-dependent kinase PR – proline rich

61. MAP Kinase Cascades Lead to Changes in Gene Expression Fig 29-5

62. TK-Linked Signal Transduction Components Receptor Non-receptor tyrosine kinases –JAKs Activate STATs (signal transducers and activators of transcription) –Src family members Src, Yes, Fyn, Lck Activated by T-cell receptors, focal adhesion kinases, and RTKs

63. T-cell-Receptor-Mediated Lymphocyte Activation Fig29-8

64. Cytokines Activate JAK-STAT Pathway Fig 29-9

65. RS/TK Signal Transduction Components Receptors –RII receptor binds ligand 1 st & phosphorylates RI upon heterodimerization –RI in turn phosphorylates smads Regulatory smads –Require phosphorylation by RI receptor –Interact with co-smad & translocate to nucleus Inhibitory smads –Bind to regulatory smads and block heterodimerization with co-smads Co-smads –Do not require phosphorylation

66. Ligands of TGF-  Superfamily Activate Smad Pathway Fig 29-10

67. Seven-Transmembrane Receptor Signaling Heterotrimeric G-proteins & Second Messenger Pathways

68. Second messengersProduction/removal Cyclic nucleotidesEnzymatic synthesis & degradation CalciumRelease from ER pumped out of cytoplasm Lipids Enzymatic synthesis & degradation Nitric oxide Enzymatic synthesis & degradation Copyright 2008 by Saunders/Elsevier. All rights reserved.

69. Second messengersMobility Cyclic nucleotidesHigh in cytoplasm CalciumLow in cytoplasm unless bound to CaM LipidsSome in membrane Some in cytoplasm Some leave the cell Nitric oxideGas diffuses locally through cells & tissues Copyright 2008 by Saunders/Elsevier. All rights reserved.

70. Second messengersTargets Cyclic nucleotidesKinases and channels CalciumCalmodulin, PKC, calpain (protease) calcium release channels, other channels LipidsPKC, seven-helix receptors Nitric oxideG-cyclase, pathogens Copyright 2008 by Saunders/Elsevier. All rights reserved.

71. 7TM Receptor Interacting with Trimeric G- Protein GAPs: RGS proteins and effectors

72. Canonical 7TM Signaling Pathway - I Activated receptor Binds heterotrimeric G-protein (G  ) –specific ones for specific receptors Activated receptor is GEF –exchanges GDP for GTP on G  –G  dissociates from G  G  activates/inhibits downstream transducers –adenylyl cyclases –phosphodiesterases –phospholipases G  activates/inhibits downstream transducers –inhibition of kinases that inactivate 7TM receptors

73. Canonical 7TM Signaling Pathway – II Adenylyl cyclase –synthesis of cAMP ([cAMP] increase 100X) –cAMP activates protein kinase A (PKA) PKA (S/T kinase) –regulated by RII (regulatory II) subunit RII subunit binds cAMP or PKA catalytic subunit –deregulation by cAMP allows PKA to phosphorylate numerous substrates –PKA can enter nucleus and phosphorylate many transcriptions factors activates and inhibits

74. Canonical 7TM Signaling Pathway – III Turning off the signals –RGS – regulators of G-protein signaling GAPs for G  subunits that stimulate GTP hydrolysis hydrolysis allows G  reassociation –downstream kinases phosphorylate 7TM receptors blocks further G protein stimulation

75. Cyclic Nucleotides

76. Adenylyl Cyclase Converts ATP to cAMP Stimulated by G s  Inhibited by G i 

77. Removal of Cyclic Nucleotides Phosphodiesterases cut 3'-O-P bond to generate 5' nucleoside monophosphates cAMP AMP cGMP GMP Phosphodiesterase

78. Cyclic Nucleotide Activated Kinases Protein kinases –PKA cAMP cAMP binds to RII subunit releases PKA from autoinhibition –PKG cGMP

79. Fig. 25-3 Protein kinase A Copyright 2008 by Saunders/Elsevier. All rights reserved.

80.  -Andrenergic Receptors

81. Cyclic nucleotide-gated channels are Na + /Ca 2+ channels gated by cAMP or cGMP binding to a cytoplasmic domain Channels gated by cyclic nucleotides Fig. 10-10 Copyright 2008 by Saunders/Elsevier. All rights reserved.

82. Odorant Receptors

83. Guanylylcyclases Fig. 24-9 Copyright 2008 by Saunders/Elsevier. All rights reserved.

84. Photoreceptors

85. Formation of Lipid 2 nd Messengers

86. Lipid Second Messengers & Targets Targets: PKC, PH-domains, IP 3 receptor, seven-helix receptors

87. Lipid 2 nd Messenger Activated Protein Kinases Protein Kinase C Family

88. Time course of lipid second messenger production Stimulation by seven-helix receptors or receptor tyrosine kinases Fig. 26-8 Copyright 2008 by Saunders/Elsevier. All rights reserved.

89. Phosphoinositol Second Messenger Signaling

90. Eicosanoids a.k.a Prostaglandins Fig. 26-9 Copyright 2008 by Saunders/Elsevier. All rights reserved.

91. Pathways of leukotriene synthesis Fig. 26-10 Targets: seven- helix receptors leading to inflammation Copyright 2008 by Saunders/Elsevier. All rights reserved.

92. Sphinogomyelinase/ ceramide signaling pathways Activator: TNF Receptor: TNFR Transducer: Sphingomyelinase Second messenger: Ceramide Targets: PKC, PP2A, protein kinase Fig. 26-11 Copyright 2008 by Saunders/Elsevier. All rights reserved.

93. Ca 2+ Second Messenger Signaling

94. Calcium release channels Fig. 26-13 Copyright 2008 by Saunders/Elsevier. All rights reserved.

95. Time course of Ca ++ release & PKC activation following activation of a Xenopus egg (mediated by IP 3 ) 0 20 40 60 80 100 120 150 seconds Fig. 26-15 Copyright 2008 by Saunders/Elsevier. All rights reserved.

96. Calcium targets Direct targetsPhysiology Ca-activated channelsSecretion, membrane excitability IP 3 receptorCa ++ release for various processes CalpainCa-dependent protease Troponin-CStriated muscle contraction Second order targets (activated by Ca-calmodulin) Calcineurin (PP2B)Lymphocyte activation Myosin-LC kinaseSmooth muscle contraction Nitric oxide synthaseNO to relax smooth muscles cAMP phosphodiesteraseDegrades cAMP CAM kinaseMulti-functional kinase Copyright 2008 by Saunders/Elsevier. All rights reserved.

97. Ca-calmodulin binding to target peptides Fig. 2-14 Copyright 2008 by Saunders/Elsevier. All rights reserved.