Receptor tyrosine kinases: regulation by

interactions and compartmentation of signaling

components

György Vereb

Experimental data included from the work of the following PhD students:

László Ujlaky-Nagy M.D.

Miklós Petrás M.D.
Árpád Szöőr M.D.

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 The 3 categories of cell surface receptors

1. Receptors without intrinsic 2. Receptors WITH intrinsic (own)

(own) enzyme activity enzyme activity
e.g. cyclases, kinases, posphatases
1a. G protein linked receptors
e.g. epinephrine, serotonin, 2a. Tyrosine kinase: EGFR,
glucagon, bradykinin R PDGFR, erbB2, Insulin R
1b. Tyrosine kinase linked 2b. Tyrosine phosphatase:
receptors leukocyte CD45
e.g. cytokine receptor
superfamily: erythropoetin, 2c. Guanylil cyclase:
interferons, interleukins atrial natriuretic factor R

1c. Receptors regulating 2d. Serine/threonine kinase:

proteolytic processes transforming growth factor  R
e.g. TNFR, Wnt/Fzd, SHH,
Delta/Notch 3. Receptors with ion channel activity
e.g. acetylcholine receptor (nicotinergic)
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 Receptor tyrosine kinases (RTK)
Epidermal growth factor (EGF) receptor
Insulin receptor
Platelet derived growth factor (PDGF) receptor

EGF receptor on
A431 cells
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 Basics of RTK activation
- the central dogma: ligand binding and dimerization

Ligand binding site

Ligand

Extracellular Ligand binding Transphosphorylation

domain dimerisation of tyrosine residues

Transmembrane
 helix
Kinase
Cytoplasmic cata-
Phospho-
domain lytic
tyrosines
site

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 Concept of domains
• 1911. P. Rous: cell-free filtrate from chicken with
sarcoma conferred the tumor to healthy chicken
• Rous sarcoma virus - 1976: src gene (SaRComa)
• Codes a small (p60) cytoplasmic tyrosine kinase

Phospho-
tyrosines

SH2: src homology domain 2

recognition of P-tyrosines
SH3: src homology domain 3 SH2 or PTB
recognition of proline rich regions domain
SH1: the tyrosine kinase domain itself

PTB: phosphotyrosine binding domain

PH: Pleckstrin homology domain
phosphoinositide binding Vereb 2013
 Role of adapter proteins
The activated receptor
binds inactive Ras-GDP
through adapter proteins:
* GRB2 (Growth factor
Receptor Binding 2) -
SH2 and SH3 domains Inactive
* Sos (Son of sevenless) - Ras-GDP Sos SH3
GRB2
proline rich regions
Sos helps Ras exchange
its GDP for GTP

Active
Ras
GTP
Sos SH3
GRB2

Signalling

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 Ras, together with Gsa, and belongs to the GTP-ase superfamily
of intracellular switch proteins
Dissociation of GDP is
promoted by guanine Cycling of Ras
nucleotide exchange Ras-GTP is formed
factors (GEF), such as Sos spontaneously

in GTP-bound, “on”
Mutants of Ras, state, Ras binds and
GEF and GAP activates effector
proteins that control
are important in
function / growth of
tumor cells
Pathogenesis

Ras is a slow GTP-ase compared to Gsα .

A GTP-ase activating protein is needed to enhance GTP hydrolysis (= GAP)
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 Activation of further enzymes by Ras
- Route to the nucleus
MAPKKK

1. Activated Ras 
Raf (serine/threonin kinase)

MAPKK
3. Activated MEK 
MAPK (mitogen activated protein kinase, Ser/Thr)
4. Further kinases, transcription factors
(TCF, SRF) are serine / threonine phosphorylated 
MEK 5. Activated transcription factor(s) bind to
DNA 
2. Activated Raf  6. Gene transcription
MEK (serine/threonine
and tyrosine kinase)
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 RTK signalling: summary of main concepts
1a. Receptor dimers, ligand binding
1b. Autophosphorylation (= transphosphorylation)
2. Protein domains specialised for recognition
SH2: src homology domain 2
PTB: phosphotyrosine binding domain
recognition of P-tyrosines
SH3: src homology domain 3
recognition of proline rich regions
PH: Pleckstrin homology domain
phosphoinositide binding
SH1: the tyrosine kinase domain itself
3. Activation of further enzymes
3a. enzyme activated through adapter proteins (SH2 and SH3)
3b. enzyme binds directly to receptor (SH2 domain)
4. Signalling cascade leading to the nucleus
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 Enzymes containing SH2 domain

Members of the Src family of protein tyrosine

kinases (source of the name)
The c-Ras associated GTPase activating protein
(rasGAP) – negative feedback
Protein phosphatase-1C (PTP1C) – negative feedback
Phosphatidylinositol-3-kinase (PI3K)
Phospholipase C-g(PLC-g
)

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 The insulin receptor
- changes in metabolism, proliferation, and survival
PTB PH
insulin
Insulin receptor PIP2 PIP3 Glut4

PDK2
PDK1
P PI3K

Akt
IRS-1
P P
P

PKCζ

glucose

GAP
uptake
mTOR GSK3
p70 S6K Rab

PP1 Glycogen
S6 synthase

Protein synthesis Bad glycogen

synthesis
survival
Source: L.M. Fletcher, Biochem. J., 2000, 352:267
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 The insulin receptor
- changes in metabolism, proliferation, and survival

insulin
Insulin receptor PIP2 PIP3 Glut4

PDK2
PDK1
P PI3K

Akt
ras SHC
IRS-1
raf P P
P
MEK PKCζ

glucose
Erk

GAP
uptake
mTOR GSK3
p70 S6K Rab
RSK

PP1 Glycogen
proliferation
S6 synthase

Protein synthesis Bad glycogen

synthesis
survival
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 IP3 and DAG as second messengers
EC space
Hormone PIP2 DAG DAG PIP2
7 TM Receptor

Cytoplasm
Protein
Phospho- kinase C Phospho-
G protein lipase Cß lipase Cγ
IP3 activated
•Ion channels receptor
IP3 receptor •Myosin LC tyrosine kinase
(Ca2+ channel)
•Glycogen synthase
SER •Ik-B
•EGFR (- feedback) Cellular response
Ca2+

Activation of
Calmodulin
dependent
enzymes

•CaM kinases (e.g. Myosin LC kinase, GPK)

•Phosphatases (calcineurin)
•cAMP phosphodiesterase

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 Calcium signal evoked by the PDGF receptor

high [Ca2+]i

0.5 IP3R

2+

Calcium (mM)
Ca

0.3

low [Ca2+]i
0.1
0 400 800
time (s) Vereb 2013
 Signal pathways of PDGFR

PDGF receptor

membrane
Tyr716 SHC-Grb2-SOS
Grb2-SOS
PLCγ Ras
IP3 ,DAG Raf
PI3K
Ca2+ Src GAP MAPKK /MEK
PKC Stat, family MAPK, ERK, JNKK
Jak
Rho, Akt / JNK
Rac PKB

cell growth motility survival mitosi cell growth

differentiation
transcriptions
mitosis

Platelet derived growth factor receptors (PDGFR) are receptor tyrosine kinases that play an
important role in proliferation and survival of tumor cells.
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 Confluence dependent expression of PDGFR-β
Receptor density inside

0,8 250
A172 Confluent
control PDGF-BB Sparse

Cell number
200
raft + SEM

0,6 2. Ab. Controll

150
Controll

0,4
100

0,2 50

0
0 10 100 1000 10000
Sparse Confluent
1,25-2x104/cm2 1-1,5x105/cm2 Fluorescence intensity
0.45
A172
0.4

Pixels above threshold

0.35

(ratio + SEM)
0.3
0.25

0.2
0.15
Expression and cell surface localization of 0.1
type beta PDGF receptors varies with 0.05
ligand stimulation and cell culture 0
confluence. Sparse Confluent
1,25-2x104/cm2 1-1,5x105/cm2Vereb 2013
 What are lipid rafts?

Lipid rafts (DRM) • detergent resistant

fraction
ec GPI-Prot • different lipid
composition
• role of cholesterol in
stability
ic
• rich in GPI linked
proteins
phospholipid • abundant in signalling
glycosphyngolipid molecules
 role in receptor
sphyngomyelin compartmentation and
regulation
cholesterol
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 PDGFR β clusters overlap with lipid rafts

A172

T98G

Lipid rafts
PDGFR clusters Overlay FITC - CTX B
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 Activation by PDGF and raft localization
Quantitative evaluation of overlap: cross-correlation
C   ( x  x )( y  y )
i j i, j i, j
Identical: C=1
  (x  x )   ( y  y)
i j i, j
2
i j i, j
2
Independent: C=0
Inverse: C= -1

control PDGF MBCD + PDGF MBCD

0.7

PDGF 0.6

0.5
C + SEM

0.4

0.3

0.2

0.1

0
PDGF + MBCD sparse confluent
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 Quantitative measurement of raft dependent PDGFR
activation
PDGFR β
GM1
P-PDGFR b
Tyr716 MAPK + (Grb2)
Tyr771 MAPK - (RasGAP)
Tyr751 PI3K
Tyr1021 PLC γ control PDGF MBCD+PDGF
Stimulation and raft dependent activation of PDGFRβ
P-PDGFR β

PPDGFR
PDGFR
Inside raft

PPDGFR
PDGFR
Outside raft
PDGFR β
Schematic pathway of quantitative
GM1 image processing
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 Raft and confluence dependent activation of Tyr 716 and 771
residues

PDGF-BB

PDGF-BB
Tyr716 Tyr771
SPARSE CONFLUENT SPARSE CONFLUENT
MAPK+ MAPK-
Grb2 RasGAP

1,8 2,5
(PPDGFR / PDGFR)Outside raft
(PPDGFR / PDGFR) Inside raft

sparse confluent sparse confluent

1,6
1,4 2
1,2
1,5
1
0,8
1
0,6
0,4 0,5
0,2
0 0
control PDGF-BB control PDGF-BB
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 MAPK mediated cell proliferation depends on cell confluence

confluent sparse
PDGF-BB
20 ng/ml 0’ 0,5’ 2’ 5’ 0’ 0,5’ 2’ 5’
pP38

pP42
Actin
Actin normalized pP38 MAPK Actin normalized pP42 MAPK
7 7
sparse confluent sparse confluent
6 6
5 5
4 4
3 3
2 2
1 1
0 0
control 0,5' PDGF- 2' PDGF-BB 5' PDGF-BB control 0,5' PDGF- 2' PDGF-BB 5' PDGF-BB
BB BB
Coherent with the microscopic results, pMAPK dependent proliferation is more activated in
dispersed cells after 20ng/ml PDGF-BB ligand stimulation.
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MAPK mediated cell proliferation depends on cell confluence

SPARSE CONFLUENT
UNSTIMULATED 2h PDGF-BB UNSTIMULATED 2h PDGF-BB
PRPPIDIUM IODIDE
Ki-67

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 Raft and confluence dependent activation of Tyr 751 residues

(PPDGFR / PDGFR)Outside raft

(PPDGFR / PDGFR) Inside raft
2
sparse confluent

1,5

PDGF-BB
Tyr751 1
SPARSE CONFLUENT
PI3K
0,5

0
control PDGF-BB
confluent sparse
Actin normalized pAkt
4
PDGF-BB sparse confluent
20 ng/ml 0 0,5 2 5 0 0,5 2 5 3,5
’ ’ ’ ’ ’ ’ ’ ’ 3

pAkt 2,5
2
Actin 1,5
1
PDGF-BB stimulation significantly increased
0,5
relative receptor phosphorylation of specific Tyr 751
0
residues and activation of Akt which could facilitate control 0,5' PDGF- 2' PDGF-BB 5' PDGF-BB
cell survival in both sparse and confluent cultures. BB Vereb 2013
 Raft and confluence dependent activation of Tyr 1021 residues

(PPDGFR / PDGFR)Outside raft

(PPDGFR / PDGFR) Inside raft
2,5
sparse confluent
2

PDGF-BB
1,5
Tyr1021
SPARSE CONFLUENT 1
PLC γ
0,5

0
control PDGF-BB
confluent sparse Actin normalized pRHOa
4,5
PDGF-BB sparse confluent
4
20 ng/ml 0 0,5 2 5 0 0,5 2 5
3,5
’ ’ ’ ’ ’ ’ ’ ’
3
pRhoA
2,5

Actin 2
1,5
Tyrosine 1021 residues were significantly
1
hyperphosphorylated following ligand stimulation
0,5
and PKC-dependent RhoA phosphorylation was
0
activated in confluent cultures. control 0,5' PDGF-BB 2' PDGF-BB 5' PDGF-BB
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PKC mediated cell migration depends on cell confluence
SPARSE CONFLUENT
2 2

Ca2+ (F340/F380)

Ca2+ (F340/F380)
1.5 1.5

1 1

0.5 0.5
0 200 400 600 0 100 200 300 400

pRhoA cortactin
PDGF-BB

PDGF-BB
SPARSE CONFLUENT SPARSE CONFLUENT

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 Redistribution of PTPases

PTPN4
Raft (GM1)
control PDGF + vanadate

0,3
SHP2 PTPN4
C + SEM

0,25

0,2

0,15

0,1

0,05

0
control PDGF+Vanadate PDGF Vanadate Vereb 2013
 Conclusions
Confluent Sparse Significance

Receptor on surface high (4x) low

Receptors in rafts more (2x) less differential function ?

Specific Tyr716
weak strong
phosphorylation
Cell proliferation
Specific Tyr771 until contact
strong weak
phosphorylation inhibition
MAPK phosphorylation weak strong
Specific Tyr751
medium strong
phosphorylation
Cell survival until
confluence reached
Akt phosphorylation medium strong

Specific Tyr1021 Cortactin

strong weak
phosphorylation redistribution and
activated migration in
RhoA phosphorylation strong weak confluent cells Vereb 2013
 Epidermal growth factor receptor (EGFR) in glia tumors

• Epidermal growth factor receptor (EGFR, ErbB1) expression is

associated with high malignity and radiation resistance
• Gain of chromosome 7 material is associated with radiation resistance
• The EGFR gene is located on chromosome 7
• Is EGFR all it takes to produce this malignant, therapy resistant
phenotype?
• … astrocytoma microcell fusions with excess chromosome 7p
material
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 Survival rate of irradiated U251 NCI clones
increases with excess amounts of chromosome 7

Excess 7p
Survival Rate

0.1

0.01
NCI
NCI+7 c5
NCI+7 c9
NCI+7 c55
0.001
0 1 2 3 4 5 6 7 8 9

Irradiation Dose (Gy)

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 In addition to EGFR, integrin b1 expression
is also elevated
600000
EGFR
Integrin b1
500000
Receptor Number ± SEM

400000

300000

200000

100000

NCI NCI+7 c5 NCI+7 c9 NCI+7 c55

excess 7p
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Interaction of EGFR and Integrins
Dimerization/aggregation

Integrin
Integrin
P PIP3
P PIP3 Ak
P P ILK
t
P
FAK

TyrK
P TyrK P PI-3K
P P

Integrin β1 is not on 7p!!!

Enhanced antiapoptosis - survival

Bcl-2 BAD
P
TF TF

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 Creating glioblastoma clones with excess
EGFR
E1H / High E1H / Low
Receptor number ± SEM (x103)

1000 EGFR
1 Integrin 100
90
800 80
70
60
600
50
40
400 30
20
10
200
0

U251 klónok expressziós szintjei 2 8 14 21 28

0
U251 U251 E1L U251 E1H/L U251 E1H/H
passage number

• transfect U251 astrocytoma with the EGFR gene only

• high (E1H) and low (E1L) expression clones
• integrin expression also increased and was stable for all clones
• a subclone similar to E1L splits off from E1H, its proportion increases with passages
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 Colony forming ability of U251 clones after irradiation
Expression levels of U251 clones
3.0 Colony forming
of view 1,200

Receptor number (x1000)

± SEM± SEM 2.5
EGFR
1,000
száma

Beta 1 integrin
2.0 U251
per field

U251 E1 Low 800

látóterenként
Kolóniák

1.5 U251 E1 High

600

1.0
Colonies

400

0.5 200

0.0 0
0 2 4 6 8 U251 E1L E1H

1
Radiation resistance
Correlation
• Higher EGFR
Túlélő hányad (± SEM)

 greater colony froming

Surviving fraction

0.1
ability
± SEM

• Higher integrin β1
0.01
 greater survival ability
Can the molecular
interactions in the background
0.001
0 2 4 6 8 be verified?
Irradiation dose (Gy) Vereb 2013
 EGFR homoassociation and EGFR - b1-integrin
heteroassociation on EGFR transfected U251
clones
EGFR homoassociation
14.0
EGFR - integrin ß1 heteroassociation
12.0
FRET % ± SEM
10.0

8.0

6.0

4.0

2.0

0.0
U251 U251 E1L U251 E1H/L U251 E1H/H

Increased EGFR and integrin β1expression is accompanied by:

• increased EGFR- integrin β1 heteroassociation
• decreased EGFR homoassociation
Average FRET% per cell, averaged over the population Vereb 2013
 Microscopic “Two-sided FRET” concept
Donor bleaching Acceptor bleaching
donor
acceptor
donor acceptor

EGFR EGF Integrin

R

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 EGFR homoassociation and EGFR-intgerin heteroassociation
are complementary at the microscopic pixel level as well
EGFR-EGFR EGFR-Integrin β1
homoassociation heteroassociation

EGFR-EGFR homoassociation
E1H / Low
E1H / High

EGFR-Integrin β1
heteroassociation
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 Akt phosphorylation upon EGF stimulus
In vitro model:
Excess EGFR induces excess integrin β1 expression
which in turn shifts signaling emphasis to integrin-EGFR
association,
with a consequential increase of EGF induced Akt
phosphorylation

change of pAkt/Akt upon EGFR

2.5
and radiation resistance Integrin
Integrin
P PIP3 2.0
P PIP3 Akt
P P ILK
P
FAK

(± SEM)
TyrK
TyrK

1.5

P P PI-3K
P P
1.0

megnövekedett túlélés, sugárrezisztencia

0.5
Akt
pAkt
0.0

U251 E1L E1H U251 U251 E1L U251 E1H

Clinical consequence?
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 EGFR and b1-integrin expression levels
on frozen intraoperative astrocytoma samples

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 EGFR - integrin b1 heteroassociation
in frozen intraoperative astrocytoma samples

EGFR integrin b1 EGFR – integrin

expression expression heteroassociation

Astrocytoma, Grade
II
(sample TC515)

Astrocytoma, Grade
IV
(sample T82)

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 EGFR - integrin b1 heteroassociation
on frozen intraoperative astrocytoma samples
12.0
II.
Mean FRET efficiency ± SEM (%)

10.0

8.0

6.0

4.0

2.0

0.0
15

16

97

26

93
B

5*

6*
3

2
0'
T3

T3

T5

T3

T5

T6

T6

T7

T7

T8
6/

0/
T8

T7

T7
T8

T1
TC

TC

TC

TC

TC
Intraoperative Tumor Samples
• Increased EGFR integrin interaction (FRET%) means worse
prognosis (single determinant in stepwise logistic regression)
• Extent of interaction is also predictive within groups
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 Survival - grade Survival - EGFR

Survival – FRET (EGFR-integrin) Survival – integrin β1

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 CONCLUSION
Functional relationship between EGFR and integrin β1 may play an important
role in tumor progression and in the development of radioresistance via the
survival-promoting PI3K/Akt pathway

This raises the possibility of therapeutically modulating EGFR-integrin and

integrin-matrix interactions

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