Program Receptors involved in the cell death. Urmas Arumäe Growth factors, receptors and inherited diseases.

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1 Program Thu Receptors involved in the cell death. Urmas Arumäe Growth factors, receptors and inherited diseases. Mart Saarma Easter week, no lectures Wed Biotechnology and medical use of growth factors. Mart Saarma Thu at VEGF family of growth factors. Note place: Biomedicum 1, Haartmanin-katu 8, lecture room 1 (ground floor). Kari Alitalo Thu 5.5. Final Exam Note place INFO2

2 Candidate Neurotrophic Factors Glial Cell Line-Derived Neurotrophic Factor (GDNF): PD, ALS, Stroke, Addiction, HD Neurturin (NRTN): PD, ALS, ALZ Artemin (ARTN): Chronic Pain Persephin (PSPN): Stroke Repair Ciliary Neurotrophic Factor (CNTF): ALZ Nerve Growth Factor (NGF): ALZ, Pain Brain-Derived Neurotrophic Factor (BDNF): Depression, HD, ALZ, ALS

3 Growth factors, receptors and diseases

4 Cytokines and interferons Old hope for the cancer treatment

5 Cytokines and neurokines Despite the lack of amino-acid-sequence similarities, Bazan first proposed a family of cytokines characterized by a four-a-helix bundle structure. These were later subclassified into short-chain and long-chain a- helix-bundle cytokines The latter subfamily comprises interleukin (IL)-6, IL-11, leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin (CT)-1, erythropoietin (Epo), granulocyte colony-stimulating factor (G-CSF), IL-12, growth hormone, prolactin, IL-10, interferon (IFN) a/b, and leptin

6 Structures of IL-6, CNTF and LIF (ribbon representation) The four long a-helices, A (red), B (green), C (yellow) and D (blue), and the connecting loops (grey), as far as they have been defined, are shown. The Brookhaven Databank accession numbers are 1IL6, 1CNT and 1LKI for IL-6, CNTF and LIF respectively.

7 Cytokines >100 local and systemic messenger molecules Involved in cell growth and differentiation repair inflammatory cascade Pro- or anti-inflammatory inflammatory IL-6 TNF IL-1 IL-10 IL-4 IL-5 Cytokine balance Imbalance of cytokines in several diseases such as RA and allergy

8 Cytokine families Interleukin-1 family IL-1, IL-1ra, IL-18 etc. Tumour Necrosis Factor family TNF, Lymphotoxin, RankL, TRANCE etc. Class I haematopoietic cytokine family IL-2, IL-4, IL-7, Erythropoietin etc. Class II haematopoietic cytokine family Interferons, IL-10 Chemokines IL-8, MIP1, RANTES, etc.

9 Cytokine receptors in disease Mutations in the receptor shared by IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 cause X-SCID (X-linked severe combined immunodeficiency) EPO receptor mutations can cause benign erythrocytosis TNF receptor mutations cause tumour necrosis factor associated periodic syndrome (TRAPS) Rank receptor mutations cause Familial expansile osteolysis (FEO). Constitutive activation of cytokine signalling pathways are associate with many leukaemias.

10 Intereferons are cytokines Type I IFNs are produced by most cell types and consist of IFN- -s and IFN- Type II IFNs are mostly secreted by lymphocytes and NK cells: INF- Synthesis of interefons is regulated by several external factors, like virus infection, dsrna etc.

11 Action of interferons

12 Signalling pathways activated by IFN- and IFN- The biological activities of IFNs are initiated by binding to their cognate receptors. This leads to the activation of receptor-associated tyrosine kinases. These kinases phosphorylate members of the STAT family of transcription factors, which can enter the nucleus and, either on their own or in combination with p48, bind to the promoters of target genes and bring about gene-specific transcriptional activation.

13 IFN- signal transduction pathway IFN Stress:osmotic shock,uv Growth factors, TGF, LPS Phosphorylation P and Recruitment of Stat1 Jak1 P P Jak2 P Ras Rac TAK1 P MKK3/6 P P p38mapk Translocation Nucleus SUMO S CBP/p300 P P GAS GAS P S727 p38mapk P Transcription

14 . Schematic representation of JAK/STAT signalling through the IL-6 and IFN- receptors

15 Effects of interefons IFNs bind to specific surface receptors on primary target cells and induce the transcription of a variety of genes that mount an antiviral response. It is characteristic of these gene products that they often depend upon viral dsrna as a co-factor in order to ensure that they are only active under conditions of infection. Thus, PKR and 2' 5' oligoadenylate synthetase (OAS) are synthesized as inactive precursors (PKRi and OASi, respectively) and are activated by dsrna (PKRa and OASa, respectively). Once activated, these gene products shut down translation.

16 Effects of interefons IFNs can also induce the synthesis of gene products that arrest the cell cycle (e.g. p21, an inhibitor of G1/S phasespecific cyclin-dependent kinases), thus blocking virus replication, or induce a pro-apoptotic state (e.g. procaspases). Finally, IFNs can induce the synthesis of proteins that are involved in the processing and presentation of virus proteins to CD8+ cytotoxic T lymphocytes (CTLs) (e.g. MHC class I proteins, components of the proteasome and peptide transporter molecules). Both types of IFN also have profound immunomodulatory effects that differ between types.

17 Transcriptional induction of the IFN- gene

18 Responses of mammalian cells to dsrna Long dsrna (>30 bp) Short interfering RNA (19-21 bp) Cleavage Activate PKR Phosphorylate eif2a Induce type I interferons (IFNs) Activate 2,5 -oligo A (2-5 A) synthetase Activate RNase L RNAi with sequencespecific effects Degradation of specific mrna Inhibition of all protein synthesis Degradation of all mrnas RNAi (Elbashir et al Nature May 2001)

19 Role of the 2-5A/RNase L system in the antiviral activity of IFNs 2'-PDE, 2',5'-phosphodiesterase; P'tase, phosphatase.

20 RNaseL mutations in different populations of prostate cancer cases aligned to the domain structure of RNase L. P-loop motifs, phosphate-binding loop motifs

21 Functional model for the activation of RNase L by 2-5A

22 Interferon & Interleukin-12 Defects Mutations in IFN RI, IFN RII, STAT1 and IL12 BCG (Bacillus Calmette Guerin) infections Infection with intracellular organisms, failure to form granulomas Mycobacterial infections due to IFN- R deficiency

23 How Erytropoetin works? Is synthesized in the kidney, relased to circulation and in the bone marrow stimulates the red blood cell proliferation. Binds to and activated Epo receptor.

24 EPO biological role EPO expressed by glial cells, neurons and endothelial cells EPO-R expressed by neurons, glial cells and cardiac myocytes, kidney cells and prostate cells EPO has neurotrophic and neuroporotective effects

25 Epo receptor activation by Epo

26

27 Receptor tyrosine kinases and oncogenes

28 Receptor tyrosine kinases

29 RTK as therapeutic targets in cancer (modified from Gschwind A, Fischer OM, Ullrich A. Nat Rev Cancer May;4(5):361-70)

30 Timeline: Breakthrough discoveries on RTK (from Gschwind A, Fischer OM, Ullrich A. Nat Rev Cancer May;4(5):361-70) 1950: Nerve Growth Factor is discovered 1980: The Epidermal Growth Factor receptor is recognized as a tyrosine kinase 1984: The Epidermal Growth Factor receptor is shown to be homologous to the retroviral v-erb-b oncogene 1987: HER2 is found to be amplified in invasive breast carcinomas 1998: Trastuzumab (Herceptin) is approved for HER2-positive breast carcinomas : Imatinib (Glivec) is approved in patients with CML (bcr-abl) and in patients with gastrointestinal stromal tumors (KIT mutated) 2003: Gefitinib (Iressa) is approved for the treatment of NSCLC (EGFR mutated) 2004: Cetuximab (EGFR) is approved in metastatic colorectal cancer 2004: Avastatin (VEGF) is approved in metastatic colorectal cancer

31 RET oncogene

32 Communication of GDNF family ligands with their receptors GDNF NRTN ARTN PSPN RET signalling signalling Saarma M, Eur. J. Biochem. 2000

33 RET structure

34 RTK activation scheme after Schlessinger et al., 2000

35 GFL RET GFR Ca 2+ TK RET9 RET51

36 RET Mutations in Human Diseases Disease Papillary Thyroid Carcinoma MEN 2A & MEN 2B Hirschsprung s Disease RET Mutations Rearrangement Point Mutations Point Mutations Frame Shift, Deletion

37 RET Ca 2+ PLC Y905 Y1015 Y1062 FRS2 Shc SOS Ras ERK Grb2

38 RET signaling pathways

39 RAS RAF MEK1 SOS ERK1/2 CREB ELK-1 raft SNT/FRS2 GRB2 SOS GDNF GFR 1 GRB2 RET py1062 SHC SHP2 GAB1/2 Cell Survival Cell Proliferation p85 AKT NF B p110 RHO FAK PI3-K RAC Lamellipodia Focal Adhesion Stress Fiber

40 GDNF receptor RET: One gene five inherited diseases Hirschsprung s disease congenital aganglionosis Ondine s curse congenital central hypoventilation Inherited cancer syndromes MEN2A MEN2B FMTC

41 The RET oncogene Different mutations Different types of cancer

42 Thyroid tumors 5/ /year Up to 60% deaths due to endocrine tumors Up to 30% general population: thyroid nodules

43 Thyroid tumors Papillary ca. (up to 80%) Follicular ca. Follicular cells Anaplastic ca. Medullary ca. (sporadic & familial: MEN 2) C-cells

44 Activating mutations in RET cause tumors in the thyroid gland and adrenal medulla Thyroid gland C-cells labelled with anti-calcitonin antibody Trachea cortex medulla Adrenal chromaffin cells Adrenal gland

45 Different mutations in RET cause distinct clinical phenotypes MEN2A FMTC P YY P FMTC MEN2A FMTC MEN2B Sporadic MTC P Y M918T RET/PTC P YY P RET/PTC rearrangement site Clinical phenotypes: MEN2A: medullary thyroid carcinoma, pheochromocytoma, parathyroid hyperplasia MEN2B: medullary thyroid carcinoma, pheochromocytoma, mucosal and gastrointestinal neuromas, skeletal abnormalities, infertility PTC: sporadic papillary thyroid carcinoma

46 Multiple Endocrine Neoplasia type 2 : MEN2A MEN2B FMTC Ca 2+ RET sporadic medullary thyroid carcinoma Cys 609 Cys 611 Cys 618 Cys 620 Cys 630 Cys 634 Glu 768 Asp Leu 790 Phe Tyr 791 Phe Val 804 Met/Leu Ala 883 Phe Ser 891 Ala Met 918 Thr

47 Papillary thyroid cancer H4 RET TK RET / PTC1 RET RFG RET TK RET / PTC3 Ca 2+ Ionizing radiations TK RET / PTC P P P P

48 Papillary Thyroid Carcinoma RET/PTC (~30%) RAS (~10%) BRAF (~50%)

49 Papillary thyroid carcinomas B-Raf

50 Normal Hirschsprung s disease

51 Hirschsprung s disease Mutations are scattered in the whole RET gene Billaud et al., 2001