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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
Signal transduction: receptor
gene
1
Intercellular communication
2
Ligand
3
Surface
receptor
Intracellular signals
cell
Intracellular
receptor
nucleus
Gene regulation
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,
- example 1
- 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)
Receptors with enzyme function
Receptors with enzyme domains
Ligands
Receptor-Tyrosine kinases
Receptor-Serine/Threonine kinases
Receptor-Tyrosine phosphatases
Receptor-Guanylyl cyclases
Cell membrane
Receptors associated with enzymes
(direct or via adaptors)
Tyrosine kinases
Serine/Threonine kinases
Phosphatases
Enzym
EnzymEnzyme
domäne
Adaptor
Kinases - Phosphorylation
Phosphatasen - Dephosphorylation
Tyrosine-OH
Tyr-Kinases
Serine-OH
Threonine-OH
Ser/Thr-Kinases
„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
(vascular endothelial cell growth factor)
Tie-R: function in angiogenesis und vessel formation
Eph-R: triggers angiogenesis, directs cell and axon migration
Ephrin receptor
Example 1
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
Blood vessels in the cornea
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
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)
Raf
MEK
ERK
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)
3 important signaling
cascades are induced:
- Ras
- PLC-g
(Phospholipase C- g)
- PI3-Kinase
(Phosphoinositol 3-Kinase)
„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
P
I
Ca++
NFAT
Calmodulin
Calcineurin
P
NFAT= transcription factor
(nuclear factor
activated T cell)
nucleus
EC “specific” factors/receptors:
VEGFR2
VEGFR1
VEGF-A, PlGF
VEGFR2
VEGF-A
VEGFR3
TIE1
TIE2
VEGF-C
ANG1,2
Y799
Y820
Src (vascular leakage)
Y925
Y936
Y951
TSAd (migration)
Y994
Y1006
Y1052
Y1057
PI-3 kinase (survival)
Y1080
Y1104
gene regulation
Y1128
Y1134
Y1175
Y1212
Y1221
Y1303
Y1307
Y1317
PLC-g
proliferation
vasculogenesis
angiogenesis
Sakurai et al.
PNAS 2005
VEGF vs. EGF signaling
PIP2
IP
VEGF
EGF
R-Tyk
R-Tyk
PLC-g
3
IP - R
3
Ras
DAG
End.Ret.
Ca
2+
PKC
2+
Ca
Raf
CAM
MEK1/2
CN
ERK1/2
NFAT
+
P
EGR-1
VEGF responsive genes
phosphorylated MAPK ERK is transprted into the nucleus,
where it phosphorylates the transcription factor TCF
or:
PLC-g
Raf
MEK
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
PI-3 Kinase Pathway and Survival
PKB, PDK:
(PKB: protein kinas B or AKT;
PDK: PI-dependenmt kinase)
Ser/Thr kinases
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:
Conective tissue,
Smooth muscle cells
basal lamina
monocellular
layer of endothelial cells
Structure of a capillary:
Endothelial cells and basal lamina, pericytes
“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 mm of a capillary
Endothelial cells respond to signals from tissue
Hypoxia
HIF
VEGF
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
Ligands and receptors
Endothelium-specific receptors:
VEGFRs: 3
Tie-Rs: 2
Ephrin-R: 1 receptor
non-specific receptors:
bFGF
PDGF
TGF-b
VEGF/VEGFR family
VEGF/VEGFR:
VEGF-A: initiation of vasculogenesis
and 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 vessels
VEGF-D: lymphatic vessels ?
VEGFR-2: growth and permeability
VEGFR-1: negative role ?, decoy receptor,
synergism with VEGFR-2 in
tumor angiogenesis
VEGFR-3: lymphatic vessels
Angiopoietins und Tie Receptors:
Ang1: remodeling and maturation
Quiescence and stability
Resistance 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 remodeling
Either regression or increased VEGF sensitivity
Ang2 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
A family of receptor serine/threonine kinases activates
transcription factors directly at receptor
Example 2
Family of factors/receptors
TGF-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)
Heterodimerisation of Type II und Type I Receptors,
Phosphorylation of SMADs
Smad 1 - 8
(Name of C.elegans/Drosophila Protein)
Transport of
phosphorylated
SMADs into nucleus
Unterlagen:
http://mailbox.univie.ac.at/erhard.hofer
Student point, Vorlesungsunterlagen
[email protected]