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 ResearchVienna Competence Center,Lazarettgasse 19, A-1090 Wien

Intercellular communication

Intracellular signals

Gene regulationLigandSurfacereceptorGenecellnucleusSignal transduction: receptor gene3211- 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 2pleiotropic 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-domneReceptors 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 membraneLigands

Kinases - PhosphorylationPhosphatasen - Dephosphorylation

Tyrosine-OHTyr-KinasesSerine-OHSer/Thr-KinasesThreonine-OHdual specificity Kinases

Families of receptor tyrosine kinasesSurface 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 AngiogenesisExample 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-g(Phospholipase C- g)

- 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-g(Phospholipase C- g)

- PI3-Kinase(Phosphoinositol 3-Kinase)

10-7 M10-3 MSecond messengerDAG, IP3 and Ca++

activated PLC-gPKCPhosphorylates many substrates,can activate MAP-kinase pathway,gene regulationCa++Calmodulin/CalcineurinNFAT- transcription factorPLC-g signaling pathway

Ca++CalmodulinCalcineurinNFATPICa++ pathway - gene regulation

The phosphatasecalcineurindephosphorylatesNFAT

NFAT translocatesinto the nucleus

NFAT= transcription factor(nuclear factoractivated T cell)nucleusP

gene regulation

proliferationvasculogenesisangiogenesis

Y799Y820Y925Y936Y951Y994Y1006Y1052Y1057Y1080Y1104Y1128Y1134Y1175Y1212Y1221Y1303Y1307Y1317Src (vascular leakage)

TSAd (migration)

PI-3 kinase (survival)

PLC-g

VEGFR2Sakurai et al.PNAS 2005EC specific factors/receptors:

VEGFR1VEGF-A, PlGFVEGFR2VEGF-AVEGFR3VEGF-CTIE1TIE2ANG1,2

VEGFPIP2Ca 2+CAMIP3-REnd.Ret.IP3NFATMEK1/2ERK1/2+CNVEGF responsive genesR-TykVEGF vs. EGF signaling Raf EGR-1R-TykEGFRas

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/proliferationor:PLC-gRafMEK

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, vasculogenesis2- 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 mm 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

HemangioblastAngioblastEC

Ligands and receptors

Endothelium-specific receptors:VEGFRs: 3Tie-Rs: 2Ephrin-R: 1 receptor

non-specific receptors:bFGFPDGFTGF-b

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/receptorsTGF-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)

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

erhard.hofer@univie.ac.at

****Scanning electron micrograph**Auswachsen von Pseudopodien, Kapillarsprosse, bildet Hohlraum, RhreEphrine Rolle bei Zusammenwachsen arterielle und vense sprouts***