1. Why signalling? Multicellular communication Nematodes (C. Elegans) Fruit Flies (Drosophila) to Man Types of cellular communication A B Direct Cell-Cell Gap Junctions Ca 2+, cAMP A B Juxacrine Cell-Cell interaction via receptors TCR: MHC NCAM:NCAM Kit:Steel (Neural crest formation) Coordinated behaviour to benefit whole organism Autocrine Massive amplification of signal IL-2 and T-cell activation

2. Paracrine Immediate environment Local, short-lived mediators (EGF, PDGF, Cytokines) Little, or no diffusion Neurones and neurotransmitters GABA, acetycholine, glycine at a high concentration

3. Endocrine Secreted Hormone [Low] =10 -8 M Binds high-affinity receptor Insulin / glucagon (Glucose metabolism) Pituitary Hormones (Metabolism / growth) Catecholamines :adrenaline, nor-adrenaline, dopamine (sympathetic nervous system) β Cell Adipocyte

4. Anatomy of a signal pathway

5. Hormone receptors

6. Classes of membrane receptors Neurotransmitters AcCh GABA Glycine Glutamate Cell adhesion and mobility

7. Signal transduction

8. Signal amplification

9. Modulation of signal pathways Saturation, specificity, competition and antagonism

10. Hormone Signalling Tyrosine Kinase Receptors Cytokine Receptors Serine Kinase Receptors G-Protein – Coupled Receptors Nuclear Receptor signalling Second Messengers: - Cyclic nucleotides - Phospholipids; Ca 2+

11. Protein Phosphorylation Dominant posttranslational protein modification Serine / Threonine (99%); Tyrosine (<1%) Protein Kinases vs Phosphatases Protein Tyrosine Kinases(PTKs): Receptor PTKs - transmembrane prot. - insulin R - EGFR,PDGFR,FGFR,NGFR Non-receptor PTKs - intracellular proteins - c-Src, JAKs,Fak etc.

12. Downloaded from: Endocrinology (on 1 February 2007 08:35 AM) The receptor protein-tyrosine kinase families Hunter et al. 1997

13. Downloaded from: Endocrinology (on 1 February 2007 08:35 AM) © 2007 Elsevier Non-receptor PTK families Hunter et al 1997

14. Downloaded from: Endocrinology (on 1 February 2007 08:35 AM) © 2007 Elsevier Role of noncatalytic segments in PDGFR signalling

15. Downloaded from: Endocrinology (on 1 February 2007 08:35 AM) © 2007 Elsevier Adaptor proteins and signalling

16. Downloaded from: Endocrinology (on 2 February 2007 03:27 PM) © 2005 Elsevier MAPK – Pathway Regulation & Function

17. Insulin Signalling Pathways Mitogenic Metabolic

18. Downloaded from: Endocrinology (on 1 February 2007 08:35 AM) © 2007 Elsevier The protein tyrosine phosphatase families Receptor-like Non-receptor like Cytoplasmic proteins eg.PTP1B, PTP-PEST

19. Growth Factors GC ERK MEK PTP + Ras Raf P P P P = Tyrosine phosphorylation to activate proteins PTP = Protein Tyrosine Phosphatase Switch activated proteins OFF by dephosphorylation MitosisERKPNucleus X Mainly work by regulating gene transcription

20. Signalling via “Cytokine Receptors” Class I (e.g.IL2R,4R,7R; GHR, PRLR, EPOR ) Class II ( e.g.IFN; IL10R ) No intrinsic enzyme activity Some cytokines may signal via MAPK / PI3K Class I/II signals through: JAK / STAT-pathway* * JAK = “ just another kinase ” / Janus kinases STAT= signal transducers and activators of transcription

21. Downloaded from: Endocrinology (on 1 February 2007 08:51 AM) © 2005 Elsevier Class I Receptors

22. Downloaded from: Endocrinology (on 1 February 2007 08:51 AM) © 2005 Elsevier JAK-STAT Signalling cytokine binding receptors dimerize JAKs approximate and transphosphorylate JAKs  receptor-P, which effect STAT docking JAKs  STAT-P STATs dimerize translocate to nucleus

23. Downloaded from: Endocrinology (on 1 February 2007 08:51 AM) © 2005 Elsevier Negative regulation of JAK-STAT signalling  PTPs e.g.SHP-1 dephosphorylate activated JAKs or receptors  Other PTPs dephosphorylate STATs  Suppressor of cytokine signalling proteins (SOCS) bind JAKs,compete with STATS for receptor docking, and target bound signalling components for proteasomal degradation  Protein inhibitor of activated STATS (PIAS) inhibit transcriptional activity of STATs

24. Signalling via Serine Kinase Receptors Prototype: TGF-β Others : Pituitary (inhibin;activin) Bone (BMPs) Sexual diff. (MIS)  Type I and II RSKs  Signalling via: Smads / Co-Smads

25. Downloaded from: Endocrinology (on 1 February 2007 08:51 AM) © 2005 Elsevier SMAD signalling  ligand binds to receptor II or II + I  heterotetrameric complex forms and  phosphorylation of receptor I, which  phosphorylates receptor regulated SMADs (R-SMAD )  R-SMAD associates with Co-SMAD, (e.g.Smad4)  translocation to nucleus  co-factors modulate  gene transcription Type I & II Receptor Serine Kinases (TGF-βR)

26. Signalling via G Protein-Coupled Receptors Class I - Rhodopsin - Adrenoceptors, Ach, Dopamine Serotonin - Angiotensin, GnRH, TSH, Vasopressin, Opioid - Somatostatin, Cannabinoid, Prostanoid Class II - Glucagon, Calcitonin, CRF, PTH, VIP Class III - Calcium sensor

27. G-protein/AC/cAMP

28. G Protein Cycle GDP/GTP exchange Dissociation Activation of effector molecules Inherent GTPase activity Reassociation βARK / PKA Phosphorylation of Receptor Ligand binds receptor p p

29. Downloaded from: Endocrinology (on 2 February 2007 03:18 PM) © 2007 Elsevier The cAMP-Dependent Signal Transduction Pathway PKA

30. Downloaded from: Endocrinology (on 2 February 2007 03:18 PM) © 2007 Elsevier Pleiotropic Actions of cAMP

31. Downloaded from: Endocrinology (on 2 February 2007 03:18 PM) © 2007 Elsevier Cross-talk of cAMP with other signalling pathways GEF=guanine nucleotide exchange factor

32. Phospholipase C PTH GαqGαq

33. Calcium as second messenger

34. Overall Signalling is essential for cellular coordination Important on a cell, tissue and whole body level Cross-talk between systems to fine-tune cell regulation Dysfunctional signalling often leads to pathology Proto-oncogene to oncogene Degeneracy in the system to compensate for loss of function Amplification and propagation of signals Signalling controls all cellular function