SIGNAL TRANSDUCTION 2) From the cell membrane to the nucleus Part B 1-TM RECEPTORS AND ASSOCIATED SIGNALLING CASCADES Erhard Hofer Department of Vascular.

SIGNAL TRANSDUCTION2) From the cell membrane to the

nucleus

Part B

1-TM RECEPTORS AND ASSOCIATED SIGNALLING CASCADES

Erhard HoferDepartment of Vascular Biology and Thrombosis Research

Vienna Competence Center,Lazarettgasse 19, A-1090 Wien

Intercellular communication

Intracellular signals

Gene regulation

Ligand

Surfacereceptor

Gene

cell

nucleus

Signal transduction: receptor gene

3

21

1- TF activation via signalling cascades2- TF activation at the receptor3- 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)

Trimeric Receptors:TNFRs (inflammation, apoptosis)

Receptors with signalling cascades including Proteolytic cleavages:WNT-R (embryonal development, adult stem cells)

Enzym-domäne

Receptors with enzyme function

Receptors with enzyme domains

Receptor-Tyrosine kinasesReceptor-Serine/Threonine kinases

Receptor-Tyrosine phosphatases Receptor-Guanylyl cyclases

Receptors associated with enzymes(direct or via adaptors)

Tyrosine kinasesSerine/Threonine kinasesPhosphatases

Cell membrane

Ligands

EnzymEnzyme

Adaptor

Kinases - Phosphorylation

Phosphatasen - Dephosphorylation

Tyrosine-OH Tyr-Kinases

Serine-OH Ser/Thr-KinasesThreonine-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 onEndothelial cells (cells of the blood vessel wall)Receptor only (mainly) expressed in endothelial cells

Induces proliferation, filopodia extension, sprouting and aSpecific function of endothelial cells, i.e. tube formation,formation of capillaries

VEGF-R Familyvascular 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 vascularizationand good blood supply for tumordissemination of metastasis via blood and lymph vessels

Blood vessels in the cornea

3 important signalingcascades are induced:

- Ras

- PLC-(Phospholipase C-

- PI3-Kinase(Phosphoinositol 3-Kinase)

Docking of proteins viaSH2 (Src-homology) domainsbind P-Tyr and neighbouring amino acidsInitially 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- MAPKKK2- MAPKK3- MAPK

MAPK: Mitogen-activated Kinase

(there are 3 main parallel MAP-Kinase cascades:MEK/ERKP38JNK)

Raf

MEK

ERK

3 important signalingcascades are induced:

- Ras

- PLC-(Phospholipase C-

- PI3-Kinase(Phosphoinositol 3-Kinase)

10-7 M

10-3 M

„Second messenger“DAG, IP3 and Ca++

activated PLC-

PKCPhosphorylates many substrates,can activate MAP-kinase pathway,gene regulation

Ca++

Calmodulin/CalcineurinNFAT- transcription factor

PLC- signaling pathway

Ca++

Calmodulin

Calcineurin

NFAT

PI

Ca++ pathway - gene regulation

The phosphatasecalcineurindephosphorylatesNFAT

NFAT translocatesinto the nucleus

NFAT= transcription factor(nuclear factoractivated T cell)

nucleus

P

gene regulation

proliferationvasculogenesisangiogenesis

Y799

Y820

Y925

Y936

Y951Y994

Y1006

Y1052Y1057

Y1080Y1104

Y1128Y1134

Y1175Y1212Y1221

Y1303Y1307

Y1317

Src (vascular leakage)

TSAd (migration)

PI-3 kinase (survival)

PLC-

VEGFR2

Sakurai et al.PNAS 2005

EC “specific” factors/receptors:

VEGFR1 VEGF-A, PlGF

VEGFR2 VEGF-AVEGFR3 VEGF-CTIE1TIE2 ANG1,2

VEGF

PIP2

Ca 2+

CAM

IP3- R

End.Ret.

IP3

NFAT

DAG

PKCCa

2+

MEK1/2

ERK1/2

+

CN

VEGF responsive genes

R-TykPLC-

VEGF vs. EGF signaling

Raf

EGR-1P

R-Tyk

EGF

Ras

phosphorylated MAPK ERK is transprted into the nucleus,where it phosphorylates the transcription factor TCF

ERK: extracellular signal regulated kinaseTCF: ternary complex factorSRF: serum response factorSRE: serum response element

(DNA binding sequence for TCF and SRF in promoter of several genes)

genes forcell cycle/proliferation

or:PLC-RafMEK

PKB, PDK:(PKB: protein kinas B or AKT;PDK: PI-dependenmt kinase)

Ser/Thr kinases

PI-3 Kinase Pathway and Survival

1- what is angiogenesis, vasculogenesis

2- receptors important for angiogenesis

Ad1) AngiogenesisFormation of capillaries by sprouting from fully differentiatedendothelial cells of the vessel wall

A small artery:Conective tissue,Smooth muscle cellsbasal laminamonocellularlayer of endothelial cells

Structure of a capillary:Endothelial cells and basal lamina, pericytes

“sprouting angiogenesis”

Sprouting of endothelial cells from differentiatedendothelial cells of the vessel wall

Wounding induces growth of capillaries:Mouse corneachemotactic response to angiogenic factors

Every cell has to be in a distance of 50 to 100 m of a capillaryEndothelial cells respond to signals from tissueHypoxiaHIFVEGF

Vasculogenesis

Formation of blood vessels by differentiation from (hem)angioblasts

Differentiation and proliferation of EC within aNon-vascularized tissue

Fromation of a primitive tubular network

Angiogenic remodeling to form vascular network

Hemangioblast Angioblast EC

Ligands and receptors

Endothelium-specific receptors:VEGFRs: 3Tie-Rs: 2

Ephrin-R: 1 receptor

non-specific receptors:bFGFPDGFTGF-

VEGF/VEGFR:VEGF-A: initiation of vasculogenesisand 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 vesselsVEGF-D: lymphatic vessels ?

VEGFR-2: growth and permeabilityVEGFR-1: negative role ?, decoy receptor,

synergism with VEGFR-2 in tumor angiogenesis

VEGFR-3: lymphatic vessels

VEGF/VEGFR family

Angiopoietins und Tie Receptors:

Ang1: remodeling and maturationQuiescence and stabilityResistance 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 remodelingEither regression or increased VEGF sensitivityAng2 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

Example 2

Family of factors/receptors

TGF- (Transforming Growth Factor-) - 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)

A family of receptor serine/threonine kinases activates transcription factors directly at receptor

Smad 1 - 8 (Name of 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.hoferStudent point, Vorlesungsunterlagen

[email protected]