Classification of Receptors
1. G Protein coupled receptors
epinephrine, serotonine, glucagon
2. Ion channel receptors
acetylcholine receptor
3. Tyrosine kinase-linked receptors
cytokine-receptor family
4. Receptors with intrinsic enzymatic activity
the receptor has intrinsic catalytic activity
receptor tyrosine kinases
Receptors with intrinsic enzymatic activity
1. Guanylatcyclase: GTP -> cGMP
ANP: peptide hormone, atrium of the heart upon
Upon rising blood pressure – decreases vascular resistance
via a cGMP dependent kinase
2. serin-threonine kinases: TGF –ß superfamily
growth inhibition, bone formation,
3. receptor tyrosine phosphatases:
CD45, expressed on B and T lymphocytes
4. RTKs: EGFR, Insulin, VEGFR
The ANP Receptor
Receptors with intrinsic enzymatic activity
1. Guanylatcyclase: GTP -> cGMP
ANP: peptide hormone, atrium of the heart upon
rising blood pressure – decreases vascular resistance
via a cGMP dependent kinase
2. serin-threonine kinases: TGF –ß superfamily
growth inhibition, bone formation,
3. receptor tyrosine phosphatases:
CD45, expressed on B and T lymphocytes
4. RTKs: EGFR, Insulin, VEGFR
CD45 exists in various isoforms
cell type specific
glykosylation
B220 B cell specific
D2: inactive
required for
D1: active phosphatase
correct folding
highly conserved
Constitutive activation of CD45 leads to
lymphoproliferation and autoimmunity in mice
Cell 2000, 103: 1059
Inactivation of CD45 leads to Severe
combined Immuno Deficiency (SCID)
The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions
to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So
far, there have been no reports to our knowledge of a human deficiency in a tyrosine-
specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a
large deletion at one allele and a point mutation at the other. The point mutation resulted
in the alteration of intervening sequence 13 donor splice site. The patient presented at 2
months of age with severe combined immunodeficiency disease. The population of
peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen
stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels
decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte
dysfunction.
Nature Med 2000, 6:343
Implications for Medicine
Inhibitors of CD45 have implications in transplant medicine –
prevention of kidney rejection in mouse models has been proven
microglial activation by ß-amyloid peptide can be prevented –
Alzheimers disease
the various specific activatio forms may allow for a relatively specific
inhibition dependent on the indication
RTKs
NGF, PDGF, FGF, EGF, Insulin
regulate cell survival, proliferation, differentiation
therefore found in cancer
constitutive active RTKs
RTKs-ras as important signalling
cascade leading to cancer
ligand binds a dimer –
dimerization of the receptor –
activation of its kinase activity –
tyrosine phosphorylation of its own
cytosolic domaine
RTKs are frequent targets in human cancer
Signalling Pathways in Cancer downstream of RTK
Sorafenib
• Hemmt Serin/Threonin- und Rezeptor-Tyrosinkinasen
• = Multikinasenhemmer
• Greift in RAS-Signasltransduktionsweg ein, indem er RAF-Kinase (=
Serin/Threonin-Kinase) hemmt verminderte Proliferation von
Tumorzellen
• Hemmt VEGF-Rezeptor keine Angiogenese keine
Nährstoffversorgung kein Wachstum
• Hemmt PDGFR (Platelet-derived growth factor)
• Hemmt c-KIT (stem-cell growth factor)
ErbB Protein Tyrosine Kinase Subfamily
Heregulin,
EGF, TNFα, NDF..
ΗΒ−ΕΓΦ
dual
cysteine
cluster
tyrosine
kinase
domain
EGFR HER4
HER2 HER3
ErbB1 ErbB4
ErbB2 ErbB3
neu
EGFR/ErbB2 Heterodimer
NH2
EGFR/ErbB1 ErbB2/HER2
membrane
Src Ras
P Tyr877
P Tyr1023 GTP Raf1
P Tyr1112 Cbl GDP
P Tyr1139
Sos
P
Grb2 MEK
Tyr1196
P Tyr1221
COOH Shc
P
Tyr1248
MAPK
Chk
Sustained MAPK activation:
G0/ G1 progression, differentiation
Herceptin efficently inhibits Her2 signalling
in breast cancer
The EGFR –
a key element in receptor cross-talk:
signal transactivation
GPCR activates metalloproteinases
The ADAMS
The IGF-receptor – a key molecule in cancer
Oncogenes that need an intact IGF-R signalling
VEGF
•VEGF is a homodimeric glycoprotein,
binding to VEGF-Receptors on
vaskular endothelial cells
•Molecular weight: 45,000Da
•VEGF plays a key role for the
formation of blood vessels
(Angiogenesis)
VEGF = vascular endothelial growth factor Ferrara N, et al. Endocr Rev 1997;18:4–25
Die VEGF Familie und ihre Rezeptoren
VEGF-A
VEGF-B VEGF-A
VEGF-C
PlGF VEGF-D
VEGFR-1 VEGFR-2
VEGFR-3
P– –P
P– –P
–P P–
P– –P
P– –P
P– –P
Migration, proliferation, permeability, DNA synthesis, survival
Angiogenesis Lymphangiogenesis
Adapted from Ferrara N. Nat Med 2003;9:669–76
Angiogenesis contributes to Tumorigenesis,
tumor growth and Metastasis
Prämalignes Maligner Tumor- Gefäß- ruhende Offene
Stadium Tumor Wachstum invasion Mikrometastase Metastasierung
(Avaskulärer (Angiogenic (Vaskularisierter (Tumorzell- (Streuung in (Zweite
Tumor) switch) Tumor) freisetzung) entfernte Organe) Angiogenese)
Schritte, bei denen Angiogenese eine Rolle bei der Tumorprogression spielt
Modifiziert nach Poon RT-P, et al. J Clin Oncol 2001;19:1207–25
The “angiogene switch”
and Tumordevelopment
Kleiner Tumor (1–2mm) größerer Tumor
• avaskulär • vaskularisiert
• ruhend • Metastasierungspotential
Angiogenic switch
führt zur Überexpression
von pro-angiogenen Faktoren,
wie zum Beispiel VEGF
Modifiziert nach Bergers G, et al. Nat Rev Cancer 2002;3:401–10
VEGF overexpression correlates
with a bad prognosis
Study Cancer n Tumours (%) Prognostic value
Gasparini, 1997 Breast 260 95 Relapse-free survival, overall survival
Toi, 1995 152 55 Increased vascular density and relapse-
free survival
Imoto, 1998 Lung NSCLC 91 53 Overall survival
O’Byrne, 2000 NSCLC 223 47 Tumour size, vascular density
Volm, 1997 SCLC 109 59 Overall survival
Maeda, 2000 GI CRC 100 37 Overall prognosis
Amaya, 1997 CRC 136 43 Vascular density
Ishigami, 1998 CRC 60 100 Clinical stage, metastasis
Ogata, 2003 Oesophagus 92 24 Overall survival
Shih, 2000 Oesophagus 117 31 Overall survival
Paley, 1997 Ovarian 68 43 Disease-free survival
Yamamoto, 1997 70 97 Overall survival
Jacobsen, 2004 Renal 229 100 Tumour size and stage, survival
Aguayo, 2002 Haem. AML 58 100 Survival
Verstovsek, 2002 Haem. CML 184 100 Survival
Antiangiogenic therapy – a double edged sword
Classification of Receptors
1. G Protein coupled receptors
epinephrine, serotonine, glucagon
2. Ion channel receptors
acetylcholine receptor
3. Tyrosine kinase-linked receptors
cytokine-receptor family
4. Receptors with intrinsic enzymatic activity
the receptor has intrinsic catalytic activity
receptor tyrosine kinases
D1 D1
D2 D2
IL-6 IL-6
IL-6D1
IL-6R D3
D2
D3
Tyrosine kinase-linked receptors
D3 D3
gp130
cytokine-receptor family
P JAK JAK P
P P
P P
SH2
STAT1/1 IL-6 target genes
STAT1/3
STAT3/3
SH2 SH2
Homo- or P P P
Hetero-Dimers Y Y
Y Y
P P P
SH2 SH2
Proliferation IL-2, -3, -4, -5, IFN-α
-7, -9, -13, -15 Growth Inhibition Immuno-
Survival IFN-β
GM-CSF, Epo, Cell cycle arrest modulation
Differentiation IFN-γ
Prl, GH, TPO Apoptosis
Cancer
Progression
P Y Jak Jak Y P
Jak1 Jak1
Jak1 P P Tyk2 Jak2
Jak2 Jak3
Stat1
Stat3 Stat2 Stat4
Stat5a
Stat5b Stat5a
Stat5b
Stat6
Cytokine signaling and specificity
O´Shea J. et al., Ann. Rheum. Dis., 2013
Hyperactive tyrosine kinase signaling
James et al. Nature. 2005;434:1144-1148
STAT5 Baxter et al. Lancet.2005;365:1054-1061
caJAK2 (V617F)
STAT5 Levine et al. Cancer Cell. 2005;7:387-397
Kralovics et al. N Engl J Med. 2005;352:1779-1790
“An acquired mutation in JAK2 has been described in
nearly all patients with the myeloproliferative
disorder (MPD), polycythemia vera (PV), and half
those with essential thrombocythemia (ET) and
idiopathic myelofibrosis (IMF). The V617F mutation
arises in a multipotent progenitor.”
The JAK2 kinase
The pseudokinase
is a Serine/Threonine kinase
Ungureanu D. et al., and
Bandaranayake RM et al., Nat.
Struc. Mol. Biol., 2011 and 2012
The correct response of the target cell to cytokines
magnitude
of response
too strong
too long
normal response
too short
too weak
duration of response
C signal new signal
C
excessive
C
C production
C C of C
mediators C C
C C C C C
C C
target cell
inefficient shut- mediator-
too many
down independent
receptors
of the signal activation
cascade (mutations)
overshooting response of the target cell possible disease development
STATs have a unique C-terminal transactivation domain
Very similar DNA binding between STATs, but tissue specific
transcriptional regulators
Kang et al., BMC Genomics, 2013
Genome-wide STAT binding validated the cytokine-dependent nature of STAT binding to DNA.
STAT binding is primarily defined by the cell type and less so by the individual STAT protein.
The number of binding sites greatly varied between different cell contexts.
The total number of STAT enriched binding sites ranged from several hundred to one hundred thousand
Overlap of common binding sites between STATs 3 and 5 in T cells exceeds binding between STAT5 in T cells
and non-T cells.
The two faces of STAT5 activation
Flt-3L OSM IL-2 IL-3 amplified / mutated
Epo
SCF IL-31 IL-4 cytokine / growth
Tpo GM-
PDGF IL-7 factor response
GH CSF
EGF IL-9 Kit (D816V)
Prl IL-5
Insulin IL-13 Flt3-ITD
IGF-1 IL-15 ErbB / EGFR
Leptin Tpo truncated G-CSFR
IL-21
TSLP Tpo-R MPL mutation
v-MPL membrane
Cytoplasm BCR-ABL p185 PY PY PI3K GAB2
BCR-ABL p210 AKT HCK,
SH2
v-ABL Y SRC
Y
TEL-JAK1/2/3 SH2
TEL-PDGFRβ caJAK2 (V617F)
FIP1L1-PDGFRα caJAK2 (R683#)
STAT5a/5a
etc. caJAK3 (A572V)
STAT5a/5b
STAT5b/5b caTYK2 (E957D)
Nucleus AR
differentiation survival
GR senescence cell cycle
ER immunity progression
PR metabolism
DNA
loop
STAT-Oligomers
pYSTAT5 as a target in myeloproliferative neoplasms
Kotecha et al. Cancer Cell, 2008 pYSTAT5 is a clinical biomarker in leukemia
But: not validated and used clinically
Hoelbl et al., EMBO Mol Med, 2010
- the BCR/ABL oncogene is addicted to STAT5, but depends not on JAK2 or STAT3
- increased expression of STAT5A/B mRNA/protein is a mechanism for imatinib drug resistance
Warsch et al., Blood, 2011
Yan et al., Blood, 2012
JAK2 V617F transformation requires STAT5 function Walz et al., Blood, 2012
Yoshimoto et al., Blood, 2009
Flt3-ITD transformation requires STAT5 function Reckzeh et al., Leukemia, 2012
Ph-like ALL requires STAT5 function Roberts et al., NEJM, 2014
JAK1: V301I, T478A/S, S512L, R532C, V623A, R629_D630del, A634D, Q644H, Y652D, Y654F, V658F, D660H,
S703I, S703G, R724H/Q, R755Q, M828T, R879S/C/H, T901R, G902E, L910P, Y999H…
JAK2: I166T, G180A, D319N, I324S, F495V, T514A/M, N533D, F537-indelsΔIREED, I540-E543delinsKK, N542-
E543del, R564Q/L, G571S/R/G, H578R, H606Q, H608N, L611V/S, V617F, C618R, D620E, L624P, R683G/K/T/S,
R867Q, D873N, P933R…
JAK3: P132T, L156P, R172W/Q, E183G, Q501H, M511T, L527P, A572V, A573V, R657Q, V722I, D846N, R887C,
I933fs, R948C, S1008*, G1101R…
TYK2: G36D, G36R, S47N, R243W, E315K, R425H, R565W, I684S, V713F, H732R, E957D, R1027H, I1112V…
Recurrent mutations in COSMIC database, many validated
culminating into STAT5 activation (or/and pYSTAT3)
Punktmutationen in STAT5A/B Transkriptionsfaktoren
76 mutations in STAT5B and 44 STAT5A
STAT5A: N522S, A630T, P636S, R649Q, D658E, R673H, R769C…
STAT5B: S552F, R566Q, W573G, G592R, G592V, G596V, N609D, N609K, R618T, T628I, R638I,
N642H, L643V, L643Q, P645S, D658G, Y665F, A714T, D727G, A729S, P736S, P736F, P736L,
Q745H… Potential C-terminal GOF mutations in COSMIC database, some validated
Mimic of cytokine signaling through STAT5 gain of function
magnitude
of respone
too strong
too long
normal
duration of response
signal
693 725 779 Onishi et al., MCB, 1998
1 136 687 Y S S 793 Moriggl et al., Cancer Cell, 2005
linker transactivation
STAT5a oligomerisation
domain cS5a DNA binding
domain domain SH2 /pY-stability
Harir et al., Blood, 2007
Li et al., Leukemia, 2007
Harir et al., Blood, 2008
S710->F Grebien et al., Blood, 2008
Baumgartner et al., Amer. J. Patho., 2010
Li et al., Blood, 2010
699 730 Li et al., Leukemia, 2010
1 136 687 Y S 786 Friedbichler et al., Blood, 2010
linker transactivation
STAT5b oligomerisation
domain cS5b DNA binding
domain domain SH2 /pY-stability
Gain of function of STAT5b in Large Granular Rajala HL et al., Blood, 2013
Lymphocytic Leukemia patients N642->H Y665->F S715->F
Hypermethylation or genetic deletion of
SOCS family members in cancer
Palmer DC and Restifo NP,
Trends Immunology, 2009
