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) Receptor Ser/Thr-Kinasen: TGF-betaRs (growth inhibition, 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 Rezeptors with enzyme domains Rezeptor-Tyrosine kinases Rezeptor-Serine/Threonine kinases Rezeptor-Tyrosine phosphatases Rezeptor-Guanylyl cyklases 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 activitz on Endothelial cells (cells of the blood vessel wall) Recptor 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 Familie 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 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)

PLC-g pathway induced by tyrosine kinase receptors VEGF PLC-g Ca++ gene regulation PKC

„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

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 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: (PDK: PI-dependenmt kinase) Ser/Thr kinases

Unterlagen: http://mailbox.univie.ac.at/erhard.hofer Student point, Vorlesungsunterlagen erhard.hofer@univie.ac.at