Signal transduction and Mechanism of Hormone action

2. Signal transduction and Mechanism of Hormone action Dr.S.Chakravarty, MD

3. Learning objectives • List the various types of receptors in the cells • Differentiate the features of water and fat soluble hormones • List the various second messengers involved in signal transduction • Describe the G protein and its coupled receptor in detail • Differentiate intrinsic and extrinsic tyrosine kinase mediated pathways of signal transduction • Describe the mechanism of insulin release and the structure of insulin receptor

5. Types of signalling

7. Receptors for various hormones • Intracellular (transcription factors) – type 1 hormones • Cell surface – type 2 hormones

8. Types of hormones based on solubility Effect Days Minutes to hours

9. Type -1 hormones –intracellular receptors

10. Type 2 hormones- have membrane receptors

13. G protein coupled receptor: • Primary receptor for most of type 2 hormones. • Link receptors to second messengers • Serpentine -7 channel transmembrane receptor • Coupled to G proteins. • 4 types of G proteins – Gs, Gi, Gq and G12 • 3 heterotrimers of G proteins – αβγ subunits.

14. GPCR – Plasma membrane coupled receptor

16. Second messenger is cAMP G – protein coupled Receptor cAMPmediated pathway Plasma Cell membrane Cytosol GTP (active) Adenylate Cyclase G-α β γ GDP (Inactive) ATP CREB P C -AMP Protein Kinase A CREB P cAMP response element binding protein CRE Enzyme Enzyme cAMP response element

17. c-AMP pathway cont.. PKA is also known as cAMP-dependent protein kinase

18. Mechanism of action of c-AMP on protein kinase-A PKA is also known as cAMP-dependent protein kinase

19. Hydrolysis of c-AMP by Phosphodiesterase H2O c-AMP 5’-AMP Phosphodiesterase Inactive Active (-) Methyl Xanthines (Caffeine, Theophylline, Theobromine)

20. Various types of G-proteins

23. Protein Phosphorylation by protein kinases • Serine / threonine phosphorylation: • Protein kinase A and C and G attach phosphate groups to hydroxyl groups of serine and threonine – to phosphorylate proteins

24. Type- 3 Second messenger as Calcium and phosphatidylinositol Calcium is required for : • Skeletal muscle contraction • Blood clotting • Secretion of various hormones and enzymes • Membrane excitability • Enzymatic activity.

26. IMP

27. Action of phospholipase C

28. Important aspect in Metabolic regulation Glucagon Epinephrine ATP ADP Protein kinase Enzyme Enzyme P H2O P Phosphatases Insulin

29. ADP RIBOSYLATION OF PROTEINS ADP-ribosyltransferases ADP-ribosyltransferase

30. ADP RIBOSYLATION OF PROTEINS • Cholera toxin– causes ADP ribosylation of GαS & subunit and inhibits intrinsic GTPase activity – prolonged action of adenylate cyclase – watery diarrhea • Pertussis toxin – causes ADP ribosylation of G α i-2 and prevents dissociation of α i-2 from βγ subunit .Gαs activity in such cells unopposed.

31. Two more toxins for USMLE ! • Diphtheria toxin – causes ADP ribosylation of eEF-2 and inhibits protein synthesis by inhibiting translocation of ribosomes • Tetrodotoxin – is a neurotoxin which acts on fast sodium channels in the neuronal membrane and inhibit action potential in the neurons.(pufferfish, porcupinefish, ocean sunfish or mola, and triggerfish, )

32. Cyclic GMP pathway ANF NO Anti anginal Drugs + membrane bound Guanylcyclase Soluble Guanyl cyclase GTP C-GMP GTP Phosphodiesterase Protein kinase G 5’-GMP Vasodilation (smooth muscle)

33. Guanyl cyclase – cGMP (class II) • Membrane bound Guanyl cyclase – Atrial natriuretic peptide (ANP). • Soluble Guanyl cyclase – nitrates, nitroprusside, NO. • Sildenafil (Viagra) – phosphodiesterase V inhibitor • Remember :-Theophylline, caffeine – phosphodiesterase inhibitors.

34. Drugs acting through NO pathway • Sodium nitroprusside – arterial and venodilator • Nitroglycerine – veins • Hydralazine – arterial • Artery – decreased TPR and BP • Vein – decreased preload and cardiac output

35. Receptor tyrosine kinase Insulin and growth factors

36. Tyrosine kinases – phosphorylate proteins at hydroxyl groups of tyrosine amino acid With intrinsic tyrosine kinase activity Without intrinsic tyrosine kinase GH, Prolactin Erythropoietin Cytokines Janus kinase STAT (signal transducers and activators of transcription) • Insulin receptor • IGF -1 • EGF, PDGF, FGF • MAP kinase  RAS protein

37. I I Alpha subunit G G G G G G G Beta subunit GLUT-4 Cell membrane PHOSPHORYLATION OF TYROSINE RESIDUES GLUT-4 Phosphorylation of Insulin Receptor substrates DIFFERENT PATHWAYS

39. One Cause for insulin resistance Serine phosphorylation instead of tyrosine phosphorylation of insulin receptor– mediated by inflammatory mediators like TNF-alpha, interleukins, free fatty acids etc.

40. Translocation of GLUT-4 receptor • Insulin dependent translocation • Well fed state/abundant glucose in blood • PI-3 kinase pathway • Exercise induced translocation: • During exercise – no insulin • AMPK pathway

42. Mechanism of action of steroid hormones Steroid hormone ( Cortisol) HSP 90 Steroid receptor SRE In the absence of the steroid hormone cortisol, Glucocorticoid receptor resides in the cytosol complexed with several chaperone proteins including Hsp90

43. Foye’s Medicinal Chemistry, 7th Edition by Lemke

44. Cytokines, TNF a, free radicals, viruses Nf-kb pathway IκB kinase Ubiquitin proteosome pathway IKKcomplex + P Ikb (Inhibitor of Κb) p50 p65 Steroids inhibit this pathway and act as potent anti-inflammatory agents Nucleus

45. Very large family of Nuclear receptor proteins DBD – DNA BINDING DOMAIN LBD – LIGAND BINDING DOMAIN HINGE REGION - Highly variable region that provides flexibility to the receptor, so it can assume different DNA-binding conformations AF-1transactivation domain referred

46. Convergence of pathways CREB binding protein & p300 coactivator

47. Function of CBP/P300 protein • Histone acetyl transferase activity • Recruitment of RNA pol II to the promoter region of the gene of interest • Helps in transcription and translation of proteins.

48. MCQ 1

49. MCQ 2

50. MCQ 3 The phosphorylase kinase-associated regulatory protein identified by the letter A in Figure 3-4 is a calcium-binding protein. Which of the following proteins represents this regulatory subunit?