2) From the cell membrane to the nucleus 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 Intracellular signals Gene regulation 2 Ligand 3 Surface receptor cell Intracellular receptor nucleus 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) - example 1 Receptor Ser/Thr-Kinasen: TGF-betaRs (growth inhibition, - 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 Receptor-Tyrosine kinases Receptor-Serine/Threonine kinases Receptor-Tyrosine phosphatases Receptor-Guanylyl cyclases Receptors associated with enzymes (direct or via adaptors) Tyrosine kinases Serine/Threonine kinases Phosphatases Ligands Cell membrane Enzym Enzyme Adaptor Enzym- domäne

Kinases - Phosphorylation Phosphatasen - Dephosphorylation Tyrosine-OH Tyr-Kinases Serine-OH Ser/Thr-Kinases Threonine-OH „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 Example 1 (vascular endothelial cell growth factor) Tie-R: function in angiogenesis und vessel formation Eph-R: triggers angiogenesis, directs cell and axon migration Ephrin receptor

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

Blood vessels in the cornea VEGF-R Family vascular endothelial cell growth factor receptor 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 Blood vessels in the cornea

Docking of proteins via SH2 (Src-homology) domains 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)

Ras activates MAP-Kinase pathway: 1- MAPKKK 2- MAPKK 3- MAPK Raf MAPK: Mitogen-activated Kinase (there are 3 main parallel MAP-Kinase cascades: MEK/ERK P38 JNK) Raf MEK ERK

3 important signaling cascades are induced: - Ras - PLC-g - PI3-Kinase (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 NFAT= transcription factor (nuclear factor activated T cell) P I Ca++ NFAT Calmodulin Calcineurin P nucleus

VEGFR2 Src (vascular leakage) TSAd (migration) PI-3 kinase (survival) EC “specific” factors/receptors: VEGFR1 VEGF-A, PlGF VEGFR2 VEGF-A VEGFR3 VEGF-C TIE1 TIE2 ANG1,2 VEGFR2 Y799 Y820 Src (vascular leakage) TSAd (migration) PI-3 kinase (survival) PLC-g Y925 Y936 Y951 Y994 Y1006 Y1052 Y1057 Y1080 Y1104 gene regulation proliferation vasculogenesis angiogenesis Y1128 Y1134 Y1175 Y1212 Y1221 Sakurai et al. PNAS 2005 Y1303 Y1307 Y1317

VEGF vs. EGF signaling VEGF responsive genes VEGF EGF PIP R-Tyk R-Tyk 2 R-Tyk R-Tyk Ras PLC-g IP 3 IP - R 3 DAG PKC Ca 2+ End.Ret. 2+ Ca Raf CAM MEK1/2 CN ERK1/2 P + EGR-1 NFAT VEGF responsive genes

phosphorylated MAPK ERK is transprted into the nucleus, where it phosphorylates the transcription factor TCF or: PLC-g Raf MEK genes for cell cycle/ proliferation 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)

PI-3 Kinase Pathway and Survival PKB, PDK: (PKB: protein kinas B or AKT; PDK: PI-dependenmt kinase) Ser/Thr kinases

2- receptors important for angiogenesis 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

layer of endothelial cells 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 Scanning electron micrograph

“sprouting angiogenesis” Sprouting of endothelial cells from differentiated endothelial cells of the vessel wall Auswachsen von Pseudopodien, Kapillarsprosse, bildet Hohlraum, Röhre Ephrine Rolle bei Zusammenwachsen arterielle und venöse sprouts

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

TGF-b (Transforming Growth Factor-b) - Receptor 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 erhard.hofer@univie.ac.at