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 erhard.hofer@univie.ac.at