1. Lecture 7 Cellular Signal Transduction Zhihong Li （李志红）

2. blotting In molecular biology and genetics, a blot is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose PVDF or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. After the blotting, the transferred proteins, DNA or RNA are then visualized by one or more different methods.

3. When environment changes: Monad——responds directly. Multicellular organisms——signal through elaborate system of intercellular or intracellular communication ， and consequently regulate functions of organisms.

4. What is Signal Transduction? Signal Transduction is the process by which a cell converts an extracellular signal into a response. Involved in: Cell-cell communication Intracellular homeostasis- internal communication Cell’s response to environment

5. Figure 15-1 Molecular Biology of the Cell (© Garland Science 2008) Generic Signalling Pathway

6. Figure 15-8 Molecular Biology of the Cell (© Garland Science 2008)

8. §1 Signaling Molecules

9. Signaling molecules Signaling molecules, which are released by signal-producing cells, reach and transfer biological signals to their target cells to initiate specific cellular responses. Signal = Ligand Ligand- A molecule that binds to a specific site on another molecule, usually a protein, ie receptor Types Extracellular molecules Intracellular molecules

10. 1. Extracellular molecules protein & peptides: Insulin, cytokine AA & its derivatives: Gly, Glu, adrenaline, thyroxine Steroid: Sex Hormone, glucocorticosteroid Fatty acid derivatives: prostaglandin(PG) Gases: Nitric Oxide (NO)

11. (1) Endocrine signal Secreted by endocrine cells. Reach target cells by blood circulation. Time of action is long. Such as insulin, thyroxine, adrenalin

12. (2) Synaptic signal (neurotransmitters) Secreted by neuronal cells. Reach another neuron by synaptic gap. Time of action is short. Such as Acetylcholine (Ach), noradrenaline

13. (3) Paracrine signaling (local chemical mediators) Secreted by common cells. Reach neighboring target cells by passive diffusion. Time of action is short. Such as GF, prostaglandin(PG)

14. (4) Autocrine signal Act back to their own cells. Such as GF, cytokine, interferon, interleukin.

15. (5) Gaseous signal Simple structure, half-life is short and active in chemistry. Such as NO, CO. GAS MOLECULE

16. GAS MOLECULE

17. 2. Intracellular molecule cAMP, cGMP nucleotides DG(diacylglycerol) lipid derivatives IP 3 (Inositol triphosphate) carbohydrate derivatives Ca 2+ ions Ras, JAK, Raf proteins

18. Second messenger: Small molecules synthesized in cells in response to an external signal are the second messengers, which are responsible for intracellular signal transduction. Such as cAMP, cGMP, DG, IP 3, Ca 2+

19. Effect by membrane receptors Effect by intracellular receptors Intracellular molecules Extracellular molecules Signal molecules cAMP, cGMP, IP 3, DG, Ca 2+ Proteins and peptides: Hormones, cytokines Amino acid derivatives: Adrenaline Fatty acid derivatives: Prostaglandins Steroid hormones, Thyroxine, VD 3

20. 花生四烯酸

21. §2 Receptor

22. Receptor Receptors are specific proteins, which are able to recognize and bind to corresponding ligand molecules, become activated, and transduce signal to next signaling molecules. Glycoprotein or Lipoprotein

23. Membrane receptors membrane Glycoprotein Intracellular receptors Cytosol or nuclei DNA binding protein

24. (1) Ligand-gate ion channels type (cyclic receptor) ligand→receptor→ion channel open or close 1. membrane receptors

25. Yellow: Leu residues Blue: polar residues

27. 1) 7-helices transmembrane receptor (2) G Protein-Coupled Receptors (serpentine R)

28. G protein refers to any protein which binds to GDP or GTP and act as signal transduction. G proteins consist of three different subunits ( , ,  -subunit).  -subunit carries GTPase activity, binding and hydrolysis of GTP. 2) G protein (Guanylate binding protein)

30.  Ｒ H     GDP  GTP   Ｇ蛋白的活化过程（演示） ACAC ATP cAMP

31. G s →  s →AC→cAMP↑ G i →  i →AC→cAMP↓ G q →  q →PI-PLC→IP 3 +DG G o →  o →ion channel 3) Classes of G protein AC: adenylate cyclase PI-PLC: phosphatidylinositol- specific phospholipase C

32. Gs and Gi

33. cAMP ATP

34. (3) Single transmembrane α-helix receptor Tyrosine protein kinase Receptor (catalytic receptor)

35. Most growth factors bind Receptor Tyrosine Kinases

36. Figure 15-53a Molecular Biology of the Cell (© Garland Science 2008)

37. Figure 15-53b Molecular Biology of the Cell (© Garland Science 2008)

39. (4) Non tyrosine protein kinase Receptor Growth Hormone R, interferon R

40. 2. Intracellular receptor (transcription regulated receptor) Intracellular R is trans-acting factor. Localized in the cytosol and/or in the nucleus. ligand: Steroid H, VD 3, Thyroxine

42. 3. Properties of binding of Ligand and Receptor highly specificity highly affinity saturation reversible binding special function model Ligand concentration Receptor saturation

43. 胞内信号级联反应 cascade

44. 4. Control of receptor activity Phosphorylation or dephosphorylation of R Enzyme catalyzed hydrolysis G protein regulation

45. §3 Pathway of Signal Transduction

46. Grey boxes: general components of signaling pathways White boxes: specific examples Ligand  Receptors  Intracellular mediators  Nuclear events Nature 411: 759 (2001)

47. Signal transduction mediated by membrane receptor cAMP dependent-protein kinase A pathway cGMP dependent PKG pathway Ca 2+ dependent PK pathway Tyrosine protein Kinase pathway NF-κB pathway TGF- βpathway

48. 1. cAMP dependent-protein kinase A pathway HR G protein Phosphorylation of Es or functional proteins Biological effects PKA cAMPAC

49. cAMP ATP AC PPi AMP PDE  磷酸二酯酶 (phosphodiesterase, PDE) 腺苷酸环化酶 (adenylate cyclase,AC) (1) cAMP metabolism

50. (2) Mechanism of cAMP effect Activate cAMP-dependent protein kinase (PKA).

52. R R cAMP 蛋白激酶 A 的激活机制（演示） Ｃ Ｃ

53. Figure 15-36 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

54. Phosphorylate specifically Ser/Thr residues in several proteins （ 1 ） Regulation of metabolism （ 2 ） Regulation of gene expression (3) PKA effect

55. Increase glycogen catabolism

56. Response elements are the recognition sites of certain transcription factors. Response ElementTranscription FactorConsensus Sequence CRECREBTGACGTCA EREEstrogen receptorAGGTCANNNTGACCT GRE Glucocorticoid receptor AGAACANNNTGTTCT HSEHeat shock factorGAANNTTCNNGAA SRESerum response factorCC(A/T) 6 GG （ 2 ） Regulation of gene expression CRE : cAMP response element (TGACGTCA) CREB: CRE binding protein

57. Figure 15-36 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008) CRE(cAMP response element); CREB: CRE binding protein

58. Ｃ Ｃ Structure gene ＣＲＥＢＣＲＥＢ ＣＲＥＢＣＲＥＢ 细胞核细胞核 Pi ＣＲＥＢＣＲＥＢ ＣＲＥＢＣＲＥＢ ＣＲＥ DNA protein

59. Figure 15-36 Molecular Biology of the Cell (© Garland Science 2008)

60. 2. Ca 2+ dependent PK pathway (1) Ca 2+ -DAG -dependent PKC pathway H R PIP 2 G protein PLC IP 3 DAG ER PKC Ca 2+ PS Biological effects Phosphorylation of Es or functional proteins

61. [Ca 2+ ] i 0.01-1  mol/L （ 10 -7 mol/L ） [Ca 2+ ] o 2.5mmol/L （ 10 -3 mol/L ） 5000~10000×

62. Three Types of Inositol phospholipids PI, PI(4)P, PI(4,5)P2

63. Phospholipase C-  (PLC-  ) Produces DAG (diacylglycerol) and IP3 (inositol 1,4,5- trisphosphate)

64. 1 ） Function of DG and IP 3 IP 3 + R→open of Ca 2 ＋ channel →[Ca 2 ＋ ]↑ PS, Ca 2 ＋ DAGPKC ↑

65. 2 ） Function of PKC Regulation of metabolism PKC →Ser/Thr-P of R, enzyme. Gene expression

66. (2) Ca 2 ＋ -CaM dependent protein kinase pathway HR G protein PLC IP 3 Ca 2+ CaM CaMK Biological effects Phosphorylation of Es or functional proteins CaM: Calmodulin

67. Calmodulin (CaM ） Ca 2 ＋ binding protein 4 Ca 2 ＋ + CaM → Ca 2 ＋ - CaM ↓ CaM kinase↑ ↓ Ser/Thr - P ↓ Ca 2 ＋ pump, AC ↑ GC ↑

69. 3. Tyrosine-protein kinase pathway (TPK) TPK receptor is related to proliferation, differentiation, dissociation, carcinomatous change. TPK ： receptor TPK ： membrane non receptor TPK ： cytosol

70. (1) Receptor TPK － Ras － MAPK pathway GRB 2, SOS, Ras, Raf Small G protein: Ras MAPK (mitogen-activated protein kinase) ： MAPK 、 MAPKK 、 MAPKKK

71. TK Intracellular Domain Transmembrane Domain Extracellular Domain Receptor TPK Structure

72. Seven subfamilies of receptor tyrosine kinases

74. TK EGFR Homo Dimerisation EGFR Stimulation & dimerisation

75. TK Hetero Dimerisation EGFR stimulation cont…

76. The importance of receptor oligomerization

77. Figure 15-54 Molecular Biology of the Cell (© Garland Science 2008) The docking of signaling molecules at RTK

78. The binding of SH2-containing intracellular signaling proteins to an activated PDGF receptor The structural view of SH2 domain SH2 domain (Scr homology 2 domain) Src: sarcoma

79. Son of Sevenless (SOS) protein is a guanine nucleotide exchange factor(GEF) that acts on Ras-GTPases.

80. The regulation of Ras activity, a famous downstream molecule of RTK responsible for cancer development Guanine nucleotide exchange factor(GEF) GTPase-activating protein (GAP)

81. The activation of Ras by RTK signaling

82. MAPK (mitogen-activated protein kinase) regulated by Ras

83. EGF, PDGF receptor TPK Ras － GTP SOS － P GRB 2 － P Raf － P MAPKK － P MAPK － P trans-acting factor expression nucleus －P－P

84. Intracellular Signaling Pathways activated by RTKs and GPCRs

85. (2) JAKs － STAT pathway ligand non TPK receptor JAKs STAT gene expression STAT: Signal transductors and activator of transcription JAK: Janus Kinase The STAT proteins regulate many aspects of cell growth, survival and differentiation.

86. Interferon Dimerization of Receptor Interferon Autophosphorylation of JAK Phosphorylation of STAT Nuclear translocation of STAT Interferon response element Expression of gene Interferons (IFNs) are proteins made and released by lymphocytes in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells

87. Figure 15-68 Molecular Biology of the Cell (© Garland Science 2008)

88. Intracellular receptor (DNA transcription regulated receptor) Ligand: –Steroid H, VD 3, Thyroxine Receptor –Cytosolic R: glycocorticosteroid H –Nuclear R: thyroxine, estrogen, androgen, progesterone

89. A model of an intracellular receptor protein HSP(heat shock protein)

91. Cellular Signal Transduction and Disease

92. diarrhea Ribosylation of Arg of G 

93. Cholera toxin

94. Normal Cell Cancerous Cell Up Regulation Mutation Consequence of proliferation of EGFR receptors

95. TK Strategies to inhibit EGFR signaling - - - - EGFR tyrosine kinase inhibitors Anti-EGFR mAbs Anti-ligand mAbs Bispecific Abs Immune effector cell ATP

96. Points Signaling molecules –Extracellular molecules: Endocrine signal, Synaptic signal, Paracrine signaling, Autocrine signal, Gaseous signal –Intracellular molecule: Second messenger Receptor –membrane receptors: Ligand-gate ion channels type, G Protein- Coupled Receptors, Tyrosine protein kinase Receptor, Non tyrosine protein kinase Receptor –Intracellular receptor: Steroid hormone receptor Pathway of Signal Transduction –cAMP dependent-protein kinase A pathway: G protein –Ca 2+ dependent PK pathway –Tyrosine protein Kinase pathway: Ras –JAKs － STAT pathway