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
Intercellular communication Intracellular signals Gene regulation
Ligand
Surface receptor
Gene
cell
nucleus
Signal transduction: receptor gene
3
2 1
1- TF activation via signalling cascades 2- TF activation at the receptor 3- TF activation by membrane soluble ligands (TF: transcription factor)
Intracellular receptor
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) Receptors with signalling cascades including proteolytic cleavages: TNFRs (inflammation, apoptosis) WNT-R (embryonal development, adult stem cells)
Enzym- domäne
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
Cell membrane
Ligands
Enzym Enzyme
Adaptor
Kinases - Phosphorylation Phosphatasen - Dephosphorylation Tyrosine-OH Tyr-Kinases Serine-OH Ser/Thr-Kinases Threonine-OH „dual specificity“ Kinases
Families of receptor tyrosine kinases
Surface receptors with enzyme domains
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
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
3 important signaling cascades are induced: („text book“ picture) - Ras - PLC-γ (Phospholipase C- γ) - 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)
Grb-2 adaptor: SH2- Domain SOS is Ras-GEF (guanine nucleotide exchange factor) Ras: GTP-binding protein (Oncogene detected in rat-sarcoma)
SOS
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)
Raf MEK ERK
3 important signaling cascades are induced: - Ras - PLC-γ (Phospholipase C- γ) - PI3-Kinase (Phosphoinositol 3-Kinase)
10-7 M
10-3 M
„Second messenger“ DAG, IP3 and Ca++
activated PLC-γ
PKC Phosphorylates many substrates, can activate MAP-kinase pathway, gene regulation
Ca++ Calmodulin/ Calcineurin NFAT- transcription factor
PLC-γ signaling pathway
Ca++
Calmodulin
Calcineurin
NFAT
P I
Ca++ pathway - gene regulation The phosphatase calcineurin dephosphorylates NFAT NFAT translocates into the nucleus NFAT= transcription factor (nuclear factor activated T cell)
nucleus
P
gene regulation proliferation vasculogenesis angiogenesis
Y799 Y820
Y925 Y936
Y951 Y994
Y1006
Y1052 Y1057 Y1080 Y1104 Y1128 Y1134
Y1175 Y1212 Y1221 Y1303 Y1307 Y1317
Src (vascular leakage) TSAd (migration) PI-3 kinase (survival) PLC-γ
VEGFR2
Sakurai et al. PNAS 2005
EC “specific” factors/receptors: VEGFR1 VEGF-A, PlGF VEGFR2 VEGF-A VEGFR3 VEGF-C TIE1 TIE2 ANG1,2
“real life” picture: VEGFR2 has 19 tyr in cytoplasmic domain, at least one third can get phosphorylated, bind different SH2-domain proteins with different affinities, e.g. Y1175 preferentially binds PLC-γ and is essential for proliferation and angiogenesis
VEGF
PIP 2
Ca 2+
CAM
IP 3 - R
End.Ret.
IP 3
NFAT
DAG
PKC
This image cannot currently be displayed.
Ca 2+
MEK1/2
ERK1/2
+
CN
VEGF responsive genes
R-Tyk PLC-γ
VEGF vs. EGF signaling
Raf
EGR-1 P
R-Tyk
EGF
Ras
Different tyr kinase receptors activate signalling cascades differentially, e.g. VEGFR activates preferentially PLC-γ, EGFR the Ras pathway
phosphorylated MAPK ERK is transprted into the nucleus, where it phosphorylates the transcription factor TCF
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
or: PLC-γ Raf MEK
PKB, PDK:
(PKB: protein kinas B or AKT; PDK: PI-dependent kinase)
Ser/Thr kinases
PI-3 Kinase Pathway and Survival
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: connective tissue, smooth muscle cells basal lamina monocellular layer of endothelial cells
Structure of a capillary: Endothelial cells and basal lamina, pericytes
Towards the end of a capillary a single endothelial cell can form a tube
“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 µm of a capillary Endothelial cells respond to signals from tissue Hypoxia HIF: hypoxia inducible factor VEGF: vascular endothelial growth factor
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
Classical model of endothelial and hematopoietic cell differentiation, Recently challenged by the finding that during development hematopoietic stem cells are generated from so-called hemogenic endothelium, placing EC upstream of HSC
Receptors important for endothelial differentiation and angiogenesis
Largely endothelium-specific receptors:
VEGFRs: 3 Tie-Rs: 2
Ephrin-R: 1 receptor
non-specific receptors: bFGFR
TGF-β-R
VEGF/VEGFR: VEGF-A: initiation of vasculogenesis and sprouting angiogenesis, Induces immature vessels, Also called vascular permeability factor, Haploid insufficiency in k.o. mice, PlGF: remodeling of adult vessels VEGF-B: heart vascularization, lipid metabolism VEGF-C: lymphatic vessels VEGF-D: lymphatic vessels VEGFR-2: growth and permeability VEGFR-1: negative role, decoy receptor in development; however synergism with VEGFR-2 in tumor angiogenesis VEGFR-3: lymphatic vessels
VEGF/VEGFR family
Angiopoietins und Tie Receptors: Ang1: remodeling and maturation Quiescence and stability Resistance to permeability, Supports interaction with other cells and matrix, Controls vessel size (VEGF rather number of vessels), Repair of damaged vessels Ang2: natural antagonist of Ang1, Overexpression similar Ang-1 k.o. or Tie-2 k.o., Destabilization signal for initiation of vascular remodeling Either regression (w/o VEGF) or increased VEGF sensitivity Ang2 is induced in tumors Ang3 and Ang4: similar Ang1 and Ang2 Tie2: binds Ang1-4 Tie1: associates with Tie-2 without ligand binding, amplifies signal
Ephrine und Eph-Rezeptors: Largest family of growth factor receptors (especially important e.g. for nervous system), 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
Example 2 Family of factors/receptors TGF-β (Transforming Growth Factor-β) - Receptor (other family members: Activins, Inhibins, Bone morphogenetic substances) pleiotropic activities dependent on cell type, frequently inhibition of proliferation, induce synthesis of extracellular matrix, Bone formation, Role for dorsal-ventral specification (embryonic development)
A family of receptor serine/threonine kinases activates transcription factors directly at the receptor
Smad 1 - 8 (Name from corresponding C.elegans/Drosophila Protein)
Heterodimerisation of Type II und Type I Receptors, Phosphorylation of SMADs
Transport of phosphorylated SMADs into nucleus
Unterlagen: http://mailbox.univie.ac.at/erhard.hofer Student point, Vorlesungsunterlagen [email protected]
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