classification of receptors - meduni wien...classification of receptors 1. g protein coupled...

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

http://circres.ahajournals.org/content/vol93/issue8/images/large/4FF5.jpeg

http://circres.ahajournals.org/content/vol93/issue8/images/large/4FF2.jpeg

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

cell type specific glykosylation

B220 B cell specific

CD45 exists in various isoforms

D1: active phosphatase highly conserved

D2: inactive required for

correct folding

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)

Nature Med 2000, 6:343

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.

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

http://www.jco.org/cgi/content/full/21/14/2787/F1

http://www.jco.org/content/vol21/issue14/images/large/lj1430452001.jpeg

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

EGF, TNFα, ΗΒ−ΕΓΦ

tyrosine kinase domain

dual cysteine cluster

Heregulin, NDF..

EGFR ErbB1 HER2

ErbB2 neu

HER3 ErbB3

HER4 ErbB4

EGFR/ErbB2 Heterodimer

P

P

P

P

P

P

P

Tyr877

Tyr1023 Tyr1112

Tyr1139

Tyr1221 Tyr1196

Tyr1248

NH2

COOH

membrane EGFR/ErbB1 ErbB2/HER2

Cbl Grb2

Shc

Chk

Src

Sos

Ras GTP

GDP Raf1

MEK

MAPK

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

http://jap.physiology.org/content/vol93/issue3/images/large/dg0921786001.jpeg

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)

Ferrara N, et al. Endocr Rev 1997;18:4–25 VEGF = vascular endothelial growth factor

Presenter

Presentation Notes

Native VEGF (or VEGF-A) is a basic, heparin-binding, homodimeric glycoprotein of 45,000 Da. This means that it is made up of two identical units. Differential expression of the human VEGF gene results in the generation of four different molecular species having 121, 165, 189 and 206 amino acids, respectively. VEGF165 is the predominant molecular species produced by a variety of normal and transformed cells. VEGF is a potent mitogen for vascular endothelial cells derived from arteries, veins and lymphatics, but has no mitogenic activity for other cell types. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25.

Die VEGF Familie und ihre Rezeptoren

Adapted from Ferrara N. Nat Med 2003;9:669–76

Migration, proliferation, permeability, DNA synthesis, survival

Lymphangiogenesis Angiogenesis

– P P– – P

P–

VEGF-A VEGF-B

PlGF

VEGFR-1

VEGF-A

VEGFR-2

VEGF-C VEGF-D

VEGFR-3

P–

P– P–

P– – P – P – P

– P

Presenter

Presentation Notes

Two types of VEGF receptors have been identified. VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR/Flk-1) bind VEGF with high affinity. Both VEGF receptor-1 and VEGF receptor-2 have seven immunoglobulin (Ig)-like domains in the extracellular domain (ECD). VEGF receptor-2 has the highest binding affinity for VEGF165.1 Another member of the receptor family with seven Ig-like domains in the ECD is VEGF receptor-3 (Flt-4). Though not a receptor for VEGF, it binds VEGF-C and -D. In the embryo, VEGF receptor-3 is expressed in angioblasts and venules. Later on in development, it becomes restricted to lymphatic endothelium, suggesting that it has a role in the regulation of lymphangiogenesis.1 Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–76.

Angiogenesis contributes to Tumorigenesis, tumor growth and Metastasis

Modifiziert nach Poon RT-P, et al. J Clin Oncol 2001;19:1207–25

Schritte, bei denen Angiogenese eine Rolle bei der Tumorprogression spielt

Prämalignes Stadium

Maligner Tumor

Tumor- Wachstum

Gefäß- invasion

ruhende Mikrometastase

Offene Metastasierung

(Avaskulärer Tumor)

(Angiogenic switch)

(Vaskularisierter Tumor)

(Tumorzell- freisetzung)

(Streuung in entfernte Organe)

(Zweite Angiogenese)

Presenter

Presentation Notes

Tumour angiogenesis has been the subject of extensive research and it is now know that angiogenesis is involved in tumour development from the initial stages of cancer formation through to the growth of distant metastases.1 Poon RT-P, Fan S-T, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol 2001;19:1207–25.

The “angiogene switch” and Tumordevelopment

Modifiziert nach Bergers G, et al. Nat Rev Cancer 2002;3:401–10

Kleiner Tumor (1–2mm) • avaskulär • ruhend

größerer Tumor • vaskularisiert • Metastasierungspotential

Angiogenic switch führt zur Überexpression von pro-angiogenen Faktoren, wie zum Beispiel VEGF

Presenter

Presentation Notes

It is generally accepted that no solid tumour can grow beyond a critical size of 1–2mm or about 106 cells without an adequate vascular supply. The angiogenic ‘switch’ is reviewed in Bergers et al.1 Tumour-associated angiogenesis can be thought to go through two phases, which are separated by the ‘angiogenic switch’. The first is an ‘avascular’ phase, in which tumours are not more than 1–2mm in diameter. These tumours remain dormant, as they are in a steady state between cell proliferation and apoptosis. Some of these small tumours will switch to the second – vascular – phase, in which the tumour grows exponentially. A classic model of the angiogenic switch is that of a pair of scales laden with pro-angiogenic molecules on one side and anti-angiogenic molecules on the other side. An imbalance of these pro- and anti-angiogenic factors triggers the ‘switch’. The onset of angiogenesis or the ‘angiogenic switch’ is a discrete step that can occur at any stage of tumour progression, dependent upon the type of tumour and its micro-environment. Induction of the switch is dependent upon the balance tipping in favour of the pro-angiogenic molecules. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003;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

Presenter

Presentation Notes

The evidence linking tumour growth and metastasis with angiogenesis is compelling. Many studies have found that patients with elevated VEGF levels have poorer prognosis than those with lower or no detectable VEGF. See below for summary of outcomes for patients with VEGF-negative versus VEGF-positive tumours in the studies listed. Gasparini: when serum VEGF >1,096pg/mg protein, probability of relapse-free and overall survival decreased with increasing VEGF levels Toi: VEGF levels significantly associated with vascular density:�microvessel density ≤50 VEGF negative 23% vs VEGF positive 5%�microvessel density 51–100 VEGF negative vs 35% VEGF positive 20%�microvessel density >100 VEGF negative 10% VEGF positive 59% Imoto: survival significantly worse in patients with VEGF-positive tumours (p=0.003) O'Byrne: high VEGF levels significantly associated with high vascular grade tumours (p=0.009); significant marker for tumour size on multivariate analysis (p<0.02); 5-year survival rates 30% for patients with high levels of VEGF vs 50% for those with low levels of VEGF Volm: high VEGF levels associated with significantly shorter median survival time (p=0.019) Maeda: patients with VEGF-positive tumours had significantly (p<0.05) worse prognosis than VEGF negative Amaya: patients with VEGF-negative tumours survived significantly longer than those with VEGF-positive tumours (p<0.05) Ishigami: VEGF overexpression associated with poorer survival (p<0.001) and greater tumour infiltration (p=0.046), incidence of liver metastasis (p<0.0001), lymph node metastasis (p=0.036) Ogata: VEGF-positive survival rate significantly lower than VEGF negative (40% vs 60% at 7 years) Shih: 4-year survival rates for VEGF positive vs VEGF negative: 32.8% vs 53.3% (p=0.04) Paley: one quarter overexpressing VEGF had no evidence of disease recurrence at follow up vs three-quarters not overexpressing VEGF (p<0.001); disease-free survival VEGF positive vs VEGF negative: 22 vs 108 months (p<0.001) Yamamoto: high VEGF meant poorer survival than weak or no VEGF (p<0.01) Jacobsen: 10-year survival rates VEGF positive vs VEGF negative: 30% vs 55% Aguayo: 50-week survival for high VEGF vs low VEGF: 37% vs 70% Verstovsek: higher VEGF levels associated with smaller spleen size (p=0.004), lower white blood cell count (p=0.0006), lower percentage of peripheral blasts (p=0.04) and shorter survival (p=0.008).

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

D3

D2

D1

IL-6R

JAK JAK

IL-6

D3 D3

D2

D1

P P

Homo- or Hetero-Dimers

P

P

Y Y

SH2

SH2

P P

P P

gp130

IL-6

D3 D3

D2

D1

D2

D3

D1 IL-6

P P

P P Y

Y

SH2

SH2

IL-6 target genes STAT1/1 STAT1/3 STAT3/3

SH2

Tyrosine kinase-linked receptors cytokine-receptor family

Y Y Jak Jak P P

P P

IFN-α IFN-β IFN-γ

IL-2, -3, -4, -5, -7, -9, -13, -15 GM-CSF, Epo, Prl, GH, TPO

Jak1

Jak3 Jak2

Stat4 Stat5a Stat5b

Immuno- modulation

Stat6

Tyk2 Stat1 Stat2

Growth Inhibition Cell cycle arrest

Apoptosis

Jak1

Proliferation Survival

Differentiation Cancer

Progression

Stat3

Stat5b Stat5a

Jak1 Jak2

O´Shea J. et al., Ann. Rheum. Dis., 2013

Cytokine signaling and specificity

“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.”

James et al. Nature. 2005;434:1144-1148 Baxter et al. Lancet.2005;365:1054-1061 Levine et al. Cancer Cell. 2005;7:387-397 Kralovics et al. N Engl J Med. 2005;352:1779-1790

caJAK2 (V617F) STAT5 STAT5

Hyperactive tyrosine kinase signaling

The pseudokinase is a Serine/Threonine kinase

Ungureanu D. et al., and Bandaranayake RM et al., Nat. Struc. Mol. Biol., 2011 and 2012

The JAK2 kinase

The correct response of the target cell to cytokines

duration of response

normal response

C

magnitude of response

signal new signal

too strong

too long

too weak

too short

C

C

C

C C

C

excessive production of mediators

target cell

C C C

C

too many receptors

C C C

C

inefficient shut-down of the signal cascade

mediator-independent activation (mutations)

overshooting response of the target cell possible disease development

C

Presenter

Presentation Notes

Falsch regulierte signle führen oft zu erkrankungen Zu viel zytokin, zu viel rezeptor, falsch funktionierende proteine (mutationen), überschwengliche (oder auch zu geringe antwort der Zelle) Zeitrahmen der aktivierung ist wichtig.

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.

STAT5a/5b

Flt-3L SCF

PDGF EGF

Insulin IGF-1 Leptin

Cytoplasm

Epo Tpo GH Prl

IL-2 IL-4 IL-7 IL-9

IL-13 IL-15 IL-21 TSLP

IL-3 GM-CSF IL-5

DNA loop

OSM IL-31

Tpo Tpo-R

Nucleus

Y

SH2 Y

SH2

STAT5a/5a

STAT5b/5b

BCR-ABL p185 BCR-ABL p210 v-ABL TEL-JAK1/2/3 TEL-PDGFRβ FIP1L1-PDGFRα etc.

amplified / mutated cytokine / growth factor response

Kit (D816V) Flt3-ITD

ErbB / EGFR truncated G-CSFR

MPL mutation v-MPL

AR GR ER PR

caJAK2 (V617F) caJAK2 (R683#) caJAK3 (A572V) caTYK2 (E957D)

STAT-Oligomers

The two faces of STAT5 activation

PY PY

membrane

HCK, SRC

GAB2 PI3K

AKT

survival cell cycle

progression

differentiation senescence

immunity metabolism

http://upload.wikimedia.org/wikipedia/en/6/6d/Detective_Comics_818_2nd_print_coverart.jpg

pYSTAT5 as a target in myeloproliferative neoplasms Kotecha et al. Cancer Cell, 2008

But: not validated and used clinically pYSTAT5 is a clinical biomarker in leukemia

Hoelbl et al., EMBO Mol Med, 2010

Yoshimoto et al., Blood, 2009 Reckzeh et al., Leukemia, 2012

Warsch et al., Blood, 2011

- 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

Flt3-ITD transformation requires STAT5 function

Yan et al., Blood, 2012 Walz et al., Blood, 2012 JAK2 V617F transformation requires STAT5 function

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)

Roberts et al., NEJM, 2014 Ph-like ALL requires STAT5 function

Potential C-terminal GOF mutations in COSMIC database, some validated

76 mutations in STAT5B and 44 STAT5A

Punktmutationen in STAT5A/B Transkriptionsfaktoren

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…

Mimic of cytokine signaling through STAT5 gain of function

magnitude of respone

too strong

too long

signal

Onishi et al., MCB, 1998 Moriggl et al., Cancer Cell, 2005 Harir et al., Blood, 2007 Li et al., Leukemia, 2007 Harir et al., Blood, 2008 Grebien et al., Blood, 2008 Baumgartner et al., Amer. J. Patho., 2010 Li et al., Blood, 2010 Li et al., Leukemia, 2010 Friedbichler et al., Blood, 2010

S710->F

Y 793 1 136

domain DNA binding SH2

693 725 779 S S

transactivation /pY-stability cS5a

duration of response

normal

STAT5a oligomerisation domain

786 Y 699 730

S

linker domain

S715->F

687

N642->H Y665->F Gain of function of STAT5b in Large Granular

Lymphocytic Leukemia patients Rajala HL et al., Blood, 2013

1 136

domain DNA binding SH2

transactivation /pY-stability cS5b STAT5b oligomerisation

domain linker

domain

687

Presenter

Presentation Notes

Falsch regulierte signle führen oft zu erkrankungen Zu viel zytokin, zu viel rezeptor, falsch funktionierende proteine (mutationen), überschwengliche (oder auch zu geringe antwort der Zelle) Zeitrahmen der aktivierung ist wichtig.

Palmer DC and Restifo NP, Trends Immunology, 2009

Hypermethylation or genetic deletion of SOCS family members in cancer

classification of receptors - meduni wien...classification of receptors 1. g protein coupled...

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