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SIGNAL TRANSDUCTION 2) From the cell membrane to the nucleus Part B 1-TM RECEPTORS AND ASSOCIATED SIGNALLING CASCADES Erhard Hofer Department of Vascular Biology and Thrombosis Research Vienna Competence Center, Lazarettgasse 19, A-1090 Wien Signal transduction: receptor gene 1 Intercellular communication 2 Ligand 3 Surface receptor Intracellular signals cell Intracellular receptor nucleus Gene regulation Gene 1- TF activation via signalling cascades 2- TF activation at the receptor 3- TF activation by membrane soluble ligands (TF: transcription factor) Receptor Tyr-Kinases: VEGFRs, Tie-Rs, EphRs (neovascularization) Receptor Ser/Thr-Kinasen: TGF-betaRs (growth inhibition, - example 1 - example 2 pleiotropic effects) Rezeptor-Guanylylcyclases: ANP-R (Salt- und water balance, relaxation) Trimeric Receptors: TNFRs (inflammation, apoptosis) Receptors with signalling cascades including Proteolytic cleavages: WNT-R (embryonal development, adult stem cells) Receptors with enzyme function Receptors with enzyme domains Ligands Receptor-Tyrosine kinases Receptor-Serine/Threonine kinases Receptor-Tyrosine phosphatases Receptor-Guanylyl cyclases Cell membrane Receptors associated with enzymes (direct or via adaptors) Tyrosine kinases Serine/Threonine kinases Phosphatases Enzym EnzymEnzyme domäne Adaptor Kinases - Phosphorylation Phosphatasen - Dephosphorylation Tyrosine-OH Tyr-Kinases Serine-OH Threonine-OH Ser/Thr-Kinases „dual specificity“ Kinases Surface receptors with enzyme domains Families of receptor tyrosine kinases Receptor tyrosine kinases: (Receptors for growth, differentiation factors and a peptide hormone) EGF-R: triggers proliferation of many different cell types (epidermal growth factor receptor) Insulin-R: triggers carbohydrate metabolism, protein synthesis IGF-R: triggers growth and survival (insulin-like growth factor receptor) NGF-R: triggers survival and growth of neurons (nerve growth factor receptor) PDGF-R: triggers survival, growth, proliferation of different cell types (platelet-derived growth factor receptor), M-CSF-R: triggers proliferation and differentiation of monocytes/macrophages (macrophage colony stimulating factor receptor) FGF-R: triggers proliferation of different cell types, triggering signal in (fibroblast growth factor receptor) embryonal development VEGF-R: triggers Angiogenesis (vascular endothelial cell growth factor) Tie-R: function in angiogenesis und vessel formation Eph-R: triggers angiogenesis, directs cell and axon migration Ephrin receptor Example 1 VEGF/VEGFR Example of growth factor receptor with specific activity on Endothelial cells (cells of the blood vessel wall) Receptor only (mainly) expressed in endothelial cells Induces proliferation, filopodia extension, sprouting and a Specific function of endothelial cells, i.e. tube formation, formation of capillaries VEGF-R Family vascular endothelial cell growth factor receptor Blood vessels in the cornea VEGFs and VEGF-Rs are important for angiogenesis (blood vessel formation by sprouting from existing vessels) and lymphangiogenesis (lymph vessel formation) Important for wound healing Tumor angiogenesis: many tumors produce VEGF, leads to high vascularization and good blood supply for tumor dissemination of metastasis via blood and lymph vessels 3 important signaling cascades are induced: - Ras - PLC-g (Phospholipase C- g) - PI3-Kinase (Phosphoinositol 3-Kinase) Docking of proteins via SH2 (Src-homology) domains bind P-Tyr and neighbouring amino acids Initially described for intracellular tyr-kinase c-Src (Oncogene of Rous Sarcoma Virus) SOS Grb-2 adaptor: SH2- Domain SOS is Ras-GEF (guanine nucleotide exchange factor) Ras: GTP-binding protein (Oncogene detected in rat-sarcoma) Raf MEK ERK Ras activates MAP-Kinase pathway: 1- MAPKKK 2- MAPKK 3- MAPK MAPK: Mitogen-activated Kinase (there are 3 main parallel MAP-Kinase cascades: MEK/ERK P38 JNK) 3 important signaling cascades are induced: - Ras - PLC-g (Phospholipase C- g) - PI3-Kinase (Phosphoinositol 3-Kinase) „Second messenger“ DAG, IP3 and Ca++ 10-3 M 10-7 M PLC-g signaling pathway activated PLC-g PKC Phosphorylates many substrates, can activate MAP-kinase pathway, gene regulation Ca++ Calmodulin/ Calcineurin NFAT- transcription factor Ca++ pathway - gene regulation The phosphatase calcineurin dephosphorylates NFAT NFAT translocates into the nucleus P I Ca++ NFAT Calmodulin Calcineurin P NFAT= transcription factor (nuclear factor activated T cell) nucleus EC “specific” factors/receptors: VEGFR2 VEGFR1 VEGF-A, PlGF VEGFR2 VEGF-A VEGFR3 TIE1 TIE2 VEGF-C ANG1,2 Y799 Y820 Src (vascular leakage) Y925 Y936 Y951 TSAd (migration) Y994 Y1006 Y1052 Y1057 PI-3 kinase (survival) Y1080 Y1104 gene regulation Y1128 Y1134 Y1175 Y1212 Y1221 Y1303 Y1307 Y1317 PLC-g proliferation vasculogenesis angiogenesis Sakurai et al. PNAS 2005 VEGF vs. EGF signaling PIP2 IP VEGF EGF R-Tyk R-Tyk PLC-g 3 IP - R 3 Ras DAG End.Ret. Ca 2+ PKC 2+ Ca Raf CAM MEK1/2 CN ERK1/2 NFAT + P EGR-1 VEGF responsive genes phosphorylated MAPK ERK is transprted into the nucleus, where it phosphorylates the transcription factor TCF or: PLC-g Raf MEK ERK: extracellular signal regulated kinase TCF: ternary complex factor SRF: serum response factor SRE: serum response element (DNA binding sequence for TCF and SRF in promoter of several genes) genes for cell cycle/ proliferation PI-3 Kinase Pathway and Survival PKB, PDK: (PKB: protein kinas B or AKT; PDK: PI-dependenmt kinase) Ser/Thr kinases 1- what is angiogenesis, vasculogenesis 2- receptors important for angiogenesis Ad1) Angiogenesis Formation of capillaries by sprouting from fully differentiated endothelial cells of the vessel wall A small artery: Conective tissue, Smooth muscle cells basal lamina monocellular layer of endothelial cells Structure of a capillary: Endothelial cells and basal lamina, pericytes “sprouting angiogenesis” Sprouting of endothelial cells from differentiated endothelial cells of the vessel wall Wounding induces growth of capillaries: Mouse cornea chemotactic response to angiogenic factors Every cell has to be in a distance of 50 to 100 mm of a capillary Endothelial cells respond to signals from tissue Hypoxia HIF VEGF Vasculogenesis Formation of blood vessels by differentiation from (hem)angioblasts Differentiation and proliferation of EC within a Non-vascularized tissue Fromation of a primitive tubular network Angiogenic remodeling to form vascular network Hemangioblast Angioblast EC Ligands and receptors Endothelium-specific receptors: VEGFRs: 3 Tie-Rs: 2 Ephrin-R: 1 receptor non-specific receptors: bFGF PDGF TGF-b VEGF/VEGFR family VEGF/VEGFR: VEGF-A: initiation of vasculogenesis and sprouting angiogenesis, Immature vessels, Vascular permeability factor, Haploid insufficiency in k.o. mice, PlGF: remodeling of adult vessels VEGF-B: heart vascularization ? VEGF-C: lymphatic vessels VEGF-D: lymphatic vessels ? VEGFR-2: growth and permeability VEGFR-1: negative role ?, decoy receptor, synergism with VEGFR-2 in tumor angiogenesis VEGFR-3: lymphatic vessels Angiopoietins und Tie Receptors: Ang1: remodeling and maturation Quiescence and stability Resistance to permeability, Supports interaction with other cells and matrix, Vessel size (VEGF number of vessels), Repair of damaged vessels Ang2: natural antagonist, Overexpression similar Ang-1 k.o. oder Tie-2 k.o., Destabilization signal for initiation of vascular remodeling Either regression or increased VEGF sensitivity Ang2 is induced in tumors Ang3: ? Ang4: ? Tie2: binds Ang1-4 Tie1: ? Ephrine und Eph-Rezeptors: Largest family of growth factor receptors, Relevant for vascular system: Ephrin B2/ Eph B4 : remodeling and maturation Different for early arterial (Ephrin B2) and venous vessels (EphB4), Hypothesis: role for fusion of arterial/ venous vessels A family of receptor serine/threonine kinases activates transcription factors directly at receptor Example 2 Family of factors/receptors TGF-b (Transforming Growth Factor-b) - Receptor (other family members: Activin, Bone morphogenetic substances) pleiotropic activities dependent on cell type, frequently inibition of proliferation, induce synthesis of extracellular matrix, Bone formation, Role for dorsal-ventral specification (embryonic development) Heterodimerisation of Type II und Type I Receptors, Phosphorylation of SMADs Smad 1 - 8 (Name of C.elegans/Drosophila Protein) Transport of phosphorylated SMADs into nucleus Unterlagen: http://mailbox.univie.ac.at/erhard.hofer Student point, Vorlesungsunterlagen [email protected]