Cell Signal Transduction and Diseases

1. Cell Signal Transduction and Diseases Jimin Shao (邵吉民) Professor, Dept. Pathology and Pathophysiology Tel: 88208209 E-mail: shaojimin@zju.edu.cn

2. Introduction: Cell signaling and Signal transduction (1) Cell signaling • (2) Types of cellular signals • (3) Functions • 2. Gap junction and diseases • (1) Structure of gap junction • (2) Function of gap junction • (3) Related diseases • 3. Receptor-Mediated Signal Transduction and diseases (1) Cell-surface receptors (2) Nuclear receptors (3) Regulatory mechanisms of signal transduction • (4) Disorders and related diseases

3. Gap Junction Introduction: Cell signaling and Signal transduction 1. Cell Signaling (1) Direct Intercellular Communication • Gap Junction (2) Signaling by plasma-membrane bound molecules (3) Receptor-Mediated Intercellular communication • Cell-Surface Receptors • Nuclear Receptors

4. 2. Types of cellular signals (1) Chemical signals Hormones, neurotransmitters, growth factors, cytokines; odor molecules; ATP, active oxygen; drugs, toxins, etc (2) Physical signals Light, electronic, mechanic, UV, heat, volume, osmotic, etc

5. (3) Modes for the function of endogenous signals Endocrine Act on a far away organ via blood circulation, seen in most hormones Paracrine Act on a nearby target, seen in GFs, CKs, etc Synaptic:Presynaptic to postsynaptic (neurotransmitters) Autocrine Act on itself after secreted, seen in GFs, especially in tumor tissues Intracrine Act on itself before secreted, seen in nuclear receptors

6. Microvesicles are membrane-enclosed extracellular vesicles shed from the plasma membrane or derived from the endosomal membrane of cells， which can mediate intercellular communication through transferring the vesicular components to recipient cells.

7. 3. Cell Signal Transduction Pathways--Physiological functions--Metabolism--Cell cycle, growth, differentiation, and apoptosis--Responses to stress--etc. LPS TNF IL-1

8. Gapjunction 1. Structure of Gap Junction • Connexins are four-pass transmembrane proteins, six of which assemble to form a channel, a connexon. • ~ 20 different isoforms of connexins in humans and mice

9. 2. Function of Gap Junction • Used by most cells in animal tissues with the exception of a few terminally differentiated cells, such as skeletal muscle cells and blood cells • Allowing inorganic ions and other small water-soluble molecules to pass directly from one cell to the other, thus coupling the cells both electrically and metabolically • In electrically excitable cells such as nerve cells allow action potential to spread rapidly without the delay that occurs at chemical synapses • The sharing of small metabolites and ions provides a mechanism for coordinating the activities of individual cells (metabolic cooperation)

10. 3. Gap Junction in Diseases • Connexin gene mutation and Diseases • Congenital non-syndromatic deafness (Cx26 mutation) • Congenital cataract (Cx50 mutation) • Axonal degeneration of peripheral nerves (Cx32 mutation) • Infertility of females (Cx37) • Gap junction and tumor promotion -- Gap junction intercellular communication down-regulated • Gap junction and embryogenesis -- embryo development (Cx43) -- nutrients transportation (Cx26) • Others

11. Receptor-Mediated Signal Transduction Systems Receptors: • Cell Surface Receptors; • Nuclear Receptors

12. Cell-surface receptors 1. Process of transmembrane signal transduction • Synthesis and secretion of signaling molecules • Receptor binding and initiation of intracellular signaling pathway • Regulation of cellular metabolism, function, gene expression, etc • Down-regulation or termination of cellular responses

13. 2. Classification of cell-surface receptors • Ion-channel-linked receptors • Involved in rapid synaptic signaling between electrically excitable cells • G-protein coupled receptors (GPCR) • Seven-pass transmembrane protein, • indirectly regulate the activity of a target protein through a trimeric GTP-binding protein (G-protein) • Enzyme-linked receptors • Single-pass transmembrane protein, • function directly as enzyme or directly associated with enzymes they activate • Others

15. Ion Channel Linked Receptor(Ligand-gated Ion Channel) 1. Classification: Class I nAchR, GABAR, 5-HT3R, GlyR Class II Glutamate/Aspartate Receptor Class III cGMP/cAMPR, IP3R, ryanodine R Class IV ATP/ADP gated channel (P2X) 2. Structure:

16. 3. Involved in rapid synaptic signaling between electrically excitable cells--A model for iGluR activation and desensitization

17. Signaling through GPCR 1. General process: GPCR (Seven-pass transmembrane protein): Adrenergic R, mAchR, opioid R, chemokine R, etc

18. G-protein: Three non-identical subunits

19. 2. GαSignaling pathways (1) Gs, Gi: • Stimulatory G protein (Gs), activates Ac; Inhibitory G protein (Gi), inhibits Ac. • cAMP is synthesized from ATP by adenylyl cyclase, and rapidly hydrolyzed by cAMP phosphodiesterase. • Usually the α subunit regulates the cyclase, although βγ complex may does so as well. • (2) Gq: Gq activates the inositol phospholipid signaling pathway by activating phospholipase C-b (PLC-b).PLC-b acts on phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] to generate: inositol (1,4,5)-triphosphate (IP3) ; diacylglycerol (DAG).

20. DAG Regulator of G-protein signaling GPCR Signaling Pathways • GPCR—GP—Ac—cAMP—PKA—CREB(CRE-binding protein) + CBP—CRE of target gene • GPCR—GP—PLCβ —IP3—Ca2+ — Calmodulin—CaM-Kinase • — DAG + Ca2+ — PKC

21. Extended reading • Classically, GPCR signaling is mediated through coupling to heterotrimeric G proteins, subsequently triggering a series of intracellular signaling cascades and ultimately leading to changes in cellular physiology. • After their activation, various GPCRs are phosphorylated by GPCR kinases (GRKs) and subsequently recruit one or both of the two isoforms of cytosolic b-arrestins (b-arrestin1 and b-arrestin2). b-Arrestin binding uncouples the receptor from the G protein, thus terminating or attenuating G protein–mediated signaling (desensitization), and facilitates clathrin-mediated endocytosis (internalization) of the receptor. • In addition to their role in the termination of G protein–mediated signaling, b-arrestins also serve as multifunctional adaptors and signal transducers, linking GPCRs to a growing list of signaling molecules, including mitogen-activated protein kinase (MAPK), the tyrosine kinase c-Src, and the Ser-Thr kinase Akt. • Whereas classical agonists stimulate both G protein–mediated and b-arrestin–mediated signaling mechanisms, “biased ligands” can selectively activate G protein or b-arrestin functions and thus elicit distinct biological effects. • Phosphorylation of GPCRs on their C termini and intracellular loops by GRKs is generally required for b-arrestin binding. • In contrast to the plethora of GPCRs, there are only seven members in the GRK family, and of those, only GRKs 2, 3, 5, and 6 are ubiquitously expressed. • Studies suggest that distinct GRKs may contribute differently to the processes of receptor desensitization, endocytosis, and signaling. • We hypothesized that the different GRKs might phosphorylate distinct sets of sites on the C terminus and internal loops of the receptor, thereby establishing a “barcode” that would instruct or determine the conformation assumed by the b-arrestin, which would, in turn, determine its functional capabilities.

22. Signaling through enzyme-linked cell-surface receptors • Receptor tyrosine kinases • Tyrosine-kinase-associated receptors • Receptor serine/threonine kinases • Receptor guanylyl cyclases • others

23. 1. Receptor tyrosine kinases • Many secreted growth factors and hormones act through receptor tyrosine kinases: • Epithelial growth factor (EGF), • platelet-derived growth factor (PDGF), • fibroblast growth factors (FGFs), • hepatocyte growth factor (HGF), • insulin, insulinlike growth factor-1 (IGF-1), • vascular endothelial growth factor (VEGF), • macrophage-colony-stimulating factor (M-CGF), • neurotrophins • Many cell-surface-bound signal proteins also act through receptor tyrosine kinases • Ephrins (Eph): regulates cell adhesion and repulsion response that guide the migration of cells and axons during development • Eph receptors: receptor tyrosine kinases • Bidirectional signaling: binding to Eph receptor can cause the activation of both Eph and Eph receptor, thus changing the behavior of both cells

24. Activated receptor tyrosine kinases phosphorylate themselves

25. (1) RAS-MAPK PathwayGF--RTK--GRB2—Ras GEF—Ras –-MAPKKK—MAPKK--MAPK (Raf—MEK1/2—ERK1/2) MAPKs:ERK JNK/SAPK p38MAPK etc

27. (2) PI3-kinase/Protein kinase B pathway • PKB (or Akt) containing a PH domain --Binds to PI(3,4,5)P3 --Activated by a phosphatidylinositol-dependent protein kinase PDK1 --Phosphorylates and inactivates BAD, a pro-apoptosis factor, promotes cell survival

28. Extended Reading: Song MS, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol. 2012;13(5):283-96. • Role of PTEN • Cell metabolism • Cell mobility and polarity • Tumor microenvironment • Cell senesence • Cancer stem cell • Nuclear function • PI3K-AKT independent functions

29. The PTEN–PI3K–AKT–mTOR pathway

31. Functions of PTEN in the nucleus

32. Regulation of PTEN • Genetic alteration • Epigenetic regulation • Post-transcriptional regulation • Post-translational modification • Protein-protein interaction

33. (3) Cross-talk between signaling pathways activated by GPCRs and RTKs

34. 2. Tyrosine-kinase-associated receptors • Cytokines --receptors --Jak-STAT signal pathway • Ligands: interferons, cytokines, etc • JAK family: JAK1-3, Tyk 2 (Just Another Kinase/Janus Kinase) (cytoplasmic tyrosine kinases) • STAT family: STAT1-7 (Signal Transducer and Activator of Transcription • Jaks phosphorylate and activate STATs, STATs then move to the nucleus and stimulate transcription • STATs contain SH2 domain and alsodock onto specific activated Receptor TK, thus being activated independent of Jaks.

36. 3. Receptor serine/threonine kinasesTGF-b superfamily--receptor serine/threonine kinases--Smads Signal Pathway • Transforming growth factor-b superfamily: TGF-bs, activins, bond morphogenetic proteins (BMPs), etc • TGF-β Receptor Superfamily: Protein serine/threonine kinase; Single pass transmembrane receptor; Type I and II • The ligand first binds to and activates type II homodimer, which recruits, phosphorylates, and activates a type I receptor dimer, forming a tetrameric receptor complex • Smads

38. 4. Receptor guanylyl cyclases • Receptor guanylyl cyclases: single pass transmembrane proteins. • Generates cGMP in response to stimuli. • cGMP binds/activates a cGMP-dependent serine/threonine protein kinase (PKG). • Atrial natriuretic peptides (ANPs) as signal molecules: regulate salt and water balance and dilate blood vessels.

39. Extended Reading: Majid S, Saini S, Dahiya R. Wnt signaling pathways in urological cancers: past decades and still growing. Mol Cancer. 2012;11(1):7.

40. Nuclear Receptors • Nuclear receptors are ligand-activated gene regulatory proteinsSteroid ReceptorsRetinoid ReceptorsOrphan Receptors Others • Some small hydrophobic signal molecules, such as steroids, thyroids, retinoids, and vitamin D, can diffuse directly across the plasma membrane to bind to nuclear receptors • Primary response • Secondary response

42. Regulatory Mechanisms of Signal transduction • Posttranslational Modification (PTM): • Phosphorylation, Acetylation, Methylation, SUMO, Ubiquitin, etc • Protein-Protein Interaction and Protein Complex: • Homodimer, Heterodimer, Polymer, etc • Protein-DNA/RNA/Lipid/small molecules, etc • Domains and signaling network: • SH2(pTyr), SH3(PXXP), PH, etc

44. Extended Reading Jason I. Herschkowitz and Xiaoyong Fu. MicroRNAs Add an Additional Layer to the Complexity of Cell Signaling. Sci. Signal.4 (184), jc5. [DOI: 10.1126/scisignal.2002182]