Buněčná signalizace – signální molekuly, membránové a nitrobuněčné receptory, signální dráhy

1. Josef Srovnal Buněčná signalizace – signální molekuly, membránové a nitrobuněčné receptory, signální dráhy Laboratoř experimentální medicíny DK LF UP a FN Olomouc

2. Cíle semináře 1. Seznámit posluchače se způsoby buněčné signalizace 2. Poukázat na její vliv na vznik lidských onemocnění. 3. Upozornit na možnosti cílené léčby.

3. Proč signalizace? Komunikace – řízení – přežití – množení – předání DNA

4. General Principles of Cell Signaling Tok informace:signalizující buňkasignální molekulyterčová (cílová) buňka

5. Signální molekuly – receptory – intracelulární - extracelulární – iontové kanály - Enzym. receptory - G protein receptory - proteinkinaza A - fosfolipaza C Signalizace - endokrinní - parakrinní - nervové - dotykové

6. Mezibuněčná signalizace volné signální molekuly vs. membránové receptory

7. Signaling moleculesIntra- and extracellular • Extracellular signaling molecules bind to either cell-surface receptors or intracellular receptors. • Most signaling molecules are hydrophilic – - unable to cross the plasma membrane – - bind to cellsurface receptors - generate signals inside the target cell. • Small signaling molecules diffuse across the plasma membrane and bind to receptors inside the target celleither in the cytosol or in the nucleus. • Many of these small signaling molecules are hydrophobic and nearly insoluble in aqueous solutions - transported bound to carrier proteins - dissociate before entering the target cell.

8. Signaling mediated by secreted molecules • Many of the same types of signaling molecules are used in paracrine, synaptic, and endocrine signaling. The crucial differences lie in the speed and selectivity with which the signals are delivered to their targets.

9. The contrast between endocrine and synaptic signaling. • Endocrine cells and nerve cells work together to coordinate the diverse activities of the billions of cells in a higher animal. • Endocrine cells secrete many different hormones into the blood to signal specific target cells. The target cells have receptors for binding specific hormones and thereby "pull" the appropriate hormones from the extracellular fluid. • In synaptic signaling the specificity arises from the contacts between nerve processes and the specific target cells they signal: usually only a target cell that is in synaptic contact with a nerve cell is exposed to the neurotransmitter released from the nerve terminal. • Whereas different endocrine cells must use different hormones in order to communicate specifically with their target cells, many nerve cells can use the same neurotransmitter and still communicate in a specific manner.

10. The same signaling molecule can induce different responses in different target cells • In some cases this is because the signaling molecule binds to different receptor proteins, as illustrated in (A) and (B). • In other cases the signaling molecule binds to identical receptor proteins that activate different response pathways in different cells, as illustrated in (B) and (C). • In all of the cases shown the signaling molecule is acetylcholine(D).

11. Signaling molecules binding to intracellular receptors • Note that all of them are small and hydrophobic. The active, hydroxylated form of vitamin D3is shown.

12. Intracellular receptor superfamily • A model of an intracellular receptor protein. In its inactive state the receptor is bound to an inhibitory protein complex that contains a heat-shock protein called Hsp90. The binding of ligand to the receptor causes the inhibitory complex to dissociate, thereby activating the receptor by exposing its DNA-binding site. • The model shown is based on the receptor for cortisol, but all of the receptors in this superfamily have a related structure.

13. Early primary response and delayed secondary response - intracellular receptor protein activation • The response to a steroid hormone is illustrated, but the same principles apply for all ligands that activate this family of receptor proteins. • Some of the primary-response proteins turn on secondary-response genes, whereas others turn off the primary-response genes.

14. Three classes of cell-surface receptors

15. Two major intracellular signaling mechanisms share common features • In both cases a signaling protein is activated by the addition of a phosphate group and inactivated by the removal of the phosphate. • In (A) the phosphate is added covalently to the signaling protein by a protein kinase; in (B) a signaling protein is induced to exchange its bound GDP for GTP. • To emphasize the similarity in the two mechanisms, ATP is shown as APP , ADP as APP, GTP as GPP , and GDP as GPP.

16. Signaling via G-Protein-linked Cell-Surface Receptors

17. A schematic drawing of a G-protein-linked receptor • Receptors that bind protein ligands have a large extracellular ligand-binding domain formed by the part of the polypeptide chain. • Receptors for small ligands such as adrenaline have small extracellular domains, and the ligand-binding site is usually deep within the plane of the membrane. • The parts of the intracellular domains that are mainly responsible for binding to trimeric G proteins are shown in orange,while those that become phosphorylated during receptor desensitization are shown in red.

18. Two major pathways by which G-protein-linked cell-surface receptors generate small intracellularmediators • In both cases the binding of an extracellular ligand alters the conformation of the cytoplasmic domain of the receptor, causing it to bind to a G protein that activates (or inactivates) a plasma membrane enzyme. • In the cyclic AMP (cAMP) pathway the enzyme directly produces cyclic AMP. • In the Ca2+ pathway the enzyme produces a soluble mediator (inositol trisphosphate) that releases Ca2+ from the endoplasmic reticulum. • Like other small intracellular mediators, both cyclic AMP and Ca2+ relay the signal by acting as allosteric effectors: they bind to specific proteins in the cell, altering their conformation and thereby their activity.

19. Cyclic AMP • The synthesis and degradation of cyclic AMP (cAMP). • It is shown as a formula, a ball-and-stick model, and as a space-filling model.

20. A current model of how Gs couples receptor activation to adenylyl cyclase activation • As long as the extracellular signaling ligand remains bound, the receptor protein can continue to activate molecules of Gs protein, thereby amplifying the response. • More important, an as can remain active and continue to stimulate a cyclase molecule for many seconds after the signaling ligand dissociates from the receptor, providing even greater amplification.

21. The activation of cyclic-AMP-dependent protein kinase (A-kinase) • The binding of cyclic AMP to the regulatory subunits induces a conformational change, causing these subunits to dissociate from the complex, thereby activating the catalytic subunits. • Each regulatory subunit has two cyclic-AMP-binding sites, and the release of the catalytic subunits requires the binding of more than two cyclic AMP molecules to the tetramer. This greatly sharpens the response of the kinase to changes in cyclic AMP concentration. • There are at least two types of A-kinase in most mammalian cells: • type I is mainly in the cytosol, whereas type II is bound via its regulatory subunit to the plasma membrane, nuclear membrane, and microtubules. In both cases, however, once the catalytic subunits are freed and active, they can migrate into the nucleus (where they can phosphorylate gene regulatory proteins), while the regulatory subunits remain in the cytoplasm.

22. The stimulation of glycogen breakdown by cyclic AMP in skeletal muscle cells. • The binding of cyclic AMP to A-kinase activates this enzyme to phosphorylate and thereby activate phosphorylase kinase, which in turn phosphorylates and activates glycogen phosphorylase, the enzyme that breaks down glycogen.

23. The role of protein phosphatase-I in the regulation of glycogen metabolism by cyclic AMP • Cyclic AMP inhibits protein phosphatase-I, which would otherwise oppose the phosphorylation reactions stimulated by cyclic AMP. It does so by activating A-kinase to phosphorylate a phosphatase inhibitor protein, which then binds to and inhibits protein phosphatase-I.

24. Target Tissue Hormone Major Response Thyroid gland thyroid-stimulating hormone (TSH) thyroid hormone synthesis and secretion Adrenal cortex adrenocorticotropic hormone (ACTH) cortisol secretion Ovary luteinizing hormone (LH) progesterone secretion Muscle adrenaline glycogen breakdown Bone parathormone bone resorption Heart adrenaline increase in heart rate and force of contraction Liver glucagon glycogen breakdown Kidney vasopressin water resorption Fat adrenaline, ACTH, glucagon, TSH triglyceride breakdown Part III. Internal Organization of the Cell Chapter 15. Cell Signaling Signaling via G-Protein-linked Cell-Surface Receptors 11 Figure 15-21. Adenylyl cyclase. In vertebrates the enzyme usually contains about 1100 amino acid residues and is thought to have two clusters of six transmembrane segments separating two similar cytoplasmic catalytic domains. There are at least six types of this form of adenylyl cyclase in mammals (types I-VI). All of them are stimulated by Gs, but type I, which is found mainly in the brain, is also stimulated by complexes of Ca2+ bound to the Ca2+-binding protein calmodulin (discussed later). Part III. Internal Organization of the Cell Chapter 15. Cell Signaling Signaling via G-Protein-linked Cell-Surface Receptors 11 Figure 15-22. Adrenaline. This hormone (also called epinephrine) is made from tyrosine and is secreted by the adrenal gland when a mammal is stressed. Some Hormone-induced Cellular Responses Mediated by Cyclic AMP • Target Tissue Hormone Major Response • Thyroid gland thyroid-stimulating h. (TSH) thyroid h synthesis and secretion • Adrenal cortex adrenocorticotropic h. (ACTH) cortisol secretion • Ovary luteinizing h. (LH) progesterone secretion • Muscle adrenaline glycogen breakdown • Bone parathormone bone resorption • Heart adrenaline heart rate and force • Liver glucagon glycogen breakdown • Kidney vasopressin water resorption • Fat adrenaline, ACTH, glucagon, TSH triglyceride breakdown

25. Inositol phospholipid pathway • The activated receptor binds to a specific trimeric G protein (Gq), causing the a subunit to dissociate and activate phospholipase C-b, which cleaves PIP2 to generate IP3 and diacylglycerol. • The diacylglycerol activates C-kinase. Both phospholipase C-b and C-kinase are water-soluble enzymes that translocate from the cytosol to the inner face of the plasma membrane in the process of being activated.

26. Inositol phospholipids (phosphoinositides) • The polyphosphoinositides (PIP and PIP2) are produced by the phosphorylation of phosphatidylinositol (PI). Although all three inositol phospholipids may be broken down in the signaling response, it is the breakdown of PIP2 that is most critical, even though it is the least abundant, constituting less than 10% of the total inositol lipids and less than 1% of the total phospholipids.

27. The hydrolysis of PIP2. • Two intracellular mediators are produced when PIP2 is hydrolyzed: • inositoltrisphosphate (IP3), which diffuses through the cytosol and releases Ca2+ from the ER, and diacylglycerol, which remains in the membrane and helps activate the enzyme proteinkinase C. • There are at least three classes of phospholipase C - b, g, and delta - and it is the b class that is activated by G-protein-linked receptors.

28. Two intracellular pathways by which activated C-kinase can activate the transcription of specific genes • In one ( red arrows) C-kinase activates a phosphorylation cascade that leads to the phosphorylation of a pivotal protein kinase called MAP-kinase, which in turn phosphorylates and activates the gene regulatory protein Elk-1. Elk-1 is bound to a short DNA sequence (called serum response element, SRE) in association with another DNA-binding protein (called serum response factor, SRF) . • In the other pathway ( green arrows) C-kinase activation leads to the phosphorylation of Ik-B, which releases the gene regulatory protein NF-kB so that it can migrate into the nucleus and activate the transcription of specific genes.

29. Some Cellular Responses Mediated by G-Protein-linked Receptors Coupled to the Inositol-PhospholipidSignaling Pathway • Target Tissue Signaling Molecule Major Response • Liver vasopressin glycogen breakdown • Pancreas acetylcholine amylase secretion • Smooth muscle acetylcholine contraction • Mast cells antigen histamine secretion • Blood platelets thrombin aggregation

30. Signaling via Enzyme-linked Cell-Surface Receptors

31. Six subfamilies of receptor tyrosine kinases • Only one or two members of each subfamily are indicated. Note that the tyrosine kinase domain is interrupted by a "kinase insert region" in some of the subfamilies. The functional significance of the cysteine-rich and immunoglobulinlike domains is unknown.

32. The serine/threonine phosphorylation cascade activated by Ras and C-kinase • In the pathway activated by receptor tyrosine kinases via Ras, the MAP-kinase-kinase-kinase is often a serine/threonine kinase called Raf, which is thought to be activated by the binding of activated Ras. • In the pathway activated by G-protein-linked receptors via C-kinase, the MAPkinase-kinase-kinase can either be Raf or a different serine/threonine kinase. • Receptor tyrosine kinases may also activate a more direct signaling pathway to the nucleus by directly phosphorylating, and thereby activating, gene regulatory proteins that contain SH2domains.

33. Growth Factors and Tumor Growth and Metastasis Metastasis • Mutations in • HER family • VEGF • MMPs • ras • p53 • COX-2 • Tumor effects • metastasis • proliferation • loss of apoptosis • infinite replication • angiogenesis • invasion Primary tumor Hanahan D, Weinberg RA. Cell. 2000;100:57-70.

34. Biologic Control of Tumor Growth Normalcells Receptors Autocrinefactors Paracrinefactors Host stromalepithelium Tumor cell

35. The HER Family of Receptors EGF TGF-a Amphiregulin Betacellulin HB-EGF Epiregulin Ligands: NRG2 NRG3 Heregulins Betacellulin Heregulins Cysteine- rich domains HER2 erbB2 neu HER3 erbB3 HER1/EGFR erbB1 HER4 erbB4 Tyrosine- kinase domains Salomon D, Brandt R, Ciardiello F, et al. Crit Rev Oncol Hematol. 1995;19:183-232. Woodburn J. Pharmacol Ther. 1999;82:241-250.

36. HER1/EGFR Dimerization HER1 homodimer HER1-HER1 Three HER1-containing heterodimers HER1-HER2 HER1-HER3 HER1-HER4 Pinkas-Kramarski R, Soussan L, Waterman H, et al. EMBO J. 1996;15:2452-2467. Klapper LN, Glathe S, Vaisman N, et al. Proc Natl Acad Sci USA. 1999;96:4995-5000.

37. Effects of HER1/EGFR Activation Extracellular Intracellular Transactivation P P Src PLCg GAP Grb2 Shc Nck Vav Grb7 Crk PKC Ras Abl JNK PI3K Akt MAPK Proliferation, invasion, metastasis, angiogenesis, and inhibition of apoptosis Woodburn J. Pharmacol Ther. 1999;82:241-250; Lynch TJ, Bell DW, Sordella R, et al. New Engl J Med. 2004;350; Knowlden JM, Hutcheson IR, Jones HE, et al. Endocrinology 2003;144:1032-1044; Chakravarti A, Chakladar A, Delaney MA, et al. Cancer Res. 2002;62:4307-4315.

38. HER1/EGFR Dysregulation in Tumors Ligand overproduction (autocrine loop) Mutations conferring constitutiveactivation Defective internalization or downregulation Overexpression P P P P P P P P P P P P EGFR vII/III EGFR vI P = phosphate De Miguel P, Royuela, Bethencourt R, et al. Cytokine. 1999;11:722-727; Normanno N, Kim N, Wen D, et al. Breast Cancer Res Treat. 1995;35:293-297; Lal A, Glazer CA, Martinson HM, et al. Cancer Res. 2002;62:3335-3339; Lynch TJ, Bell DW, Sordella R, et al. New Engl J Med. 2004;350; Paez JG, Janne PA, Lee JC, et al. Science, 29 April 2004 (10.1126/science.1099314); Yang H, Jiang D, Li W, et al. Oncogene 2000;19:1901-1914.

39. Tumors with HER1/EGFR Dysregulation Glioma Head and neck Breast Lung Esophagus Renal Gastric Colorectal Pancreatic Ovarian Bladder Cervical Prostate Salomon DS, Brandt R, Ciardiello F, et al. Crit Rev Oncol Hematol. 1995;19:183-232.Woodburn JR. Pharmacol Ther. 1999;82:241-250.

40. Common Approaches toTargeting HER1/EGFR P P P TK inhibitors Ligand– toxin conjugates Anti-ligand- blocking antibodies P Antibody– toxin conjugates Anti-HER1/EGFR-blocking antibodies Slamon DJ, Leyland-Jones B, Shak S, et al. N Engl J Med. 2001;344:783-792; Mendelsohn J, Baselga J. Oncogene. 2000;19:6550-6565; Noonberg SB, Benz CC. Drugs. 2000;59:753-767; Raymond E, Faivre S, Armann JP. Drugs. 2000;60(Suppl 1):15-23; Arteaga C. J Clin Oncol. 2001;19:32s-40s; Pedersen MW, Meltom M, Damstrup L, et al. Ann Oncol. 2001;12:745-760.

41. EGFR inhibitory Monoklonální protilátky Tyrosinkinázové inhibitory ¯ Proliferation ­ Apoptosis K-ras ­ Sensitivity to radiotherapy ¯ Invasion ¯ Metastasis ¯ Adhesion ¯ Angiogenesis Etessami A, Bourhis J. Drugs Fut. 2000;25:895-899. Moyer J, Barbacci EG, Iwata KK, et al. Cancer Res. 1997;57:4838-4848.Harari PM, Huang SM. Semin Radiat Oncol. 2002;12(Suppl 2):21-26.

42. EGFR inhibitory vs. mutace K-ras Monoklonální protilátky Tyrosinkinázové inhibitory ¯ Proliferation K-ras ­ Apoptosis ­ Sensitivity to radiotherapy ¯ Invasion ¯ Metastasis ¯ Adhesion ¯ Angiogenesis Etessami A, Bourhis J. Drugs Fut. 2000;25:895-899. Moyer J, Barbacci EG, Iwata KK, et al. Cancer Res. 1997;57:4838-4848.Harari PM, Huang SM. Semin Radiat Oncol. 2002;12(Suppl 2):21-26.

43. Onkogen K-ras • Onkogen z rodiny ras – K-ras, N-ras, H-ras • Umístění na 12 chromosomu v pozici 12.1 • Protein s GTPázovou aktivitou o velikosti 21 kDa • Součást MAPK signální dráhy - účastní se přenosu signálu z vnějšího prostředí buňky do jádra a za fyziologických podmínek je aktivovaný receptorovými tyrosinkinázami, např. EGFR1

44. K-ras v onkologii • Aktivační mutace K-ras genu v kodonu 12 a 13 • Nová moderní cílená protinádorová léčiva • EGFR inhibitory • nízkomolekulární inhibitory tyrosinkináz (Gefitinib, Erlotinib) • anti-EGFR monoklonální protilátky (Cetuximab, Vectibix) • Nemalobuněčný karcinom plic (NSCLC), skvamocelulární karcinom hlavy a krku (HNSCC) a metastazující kolorektální karcinom (CrC) • Kompletní či parciální remise je zaznamenána jen u relativně malého počtu pacientů (přibližně 10 %) • Individualizace terapie – „Správnému pacientovi, správná léčba“

45. Biologická léčba Cílená individualizovaná léčba – targeting therapy – zasahuje specifický cíl – často signální molekuly nebo receptory zodpovědné za vznik onemocnění – důležitá znalost signálních drah – základní výzkum je předpokladem pro objevování novějších a účinnějších léčiv. Prediktivní faktory – negativní a pozitivní – predikují účinnost zvoleného léčiva u konkrétního pacienta.

46. MUDr. Josef Srovnal Laboratoř experimentální medicíny DK FN a LF UP Olomouc Tel: +420 585 853 225 Email: josef.srovnal@seznam.cz www.lem.ocol.cz Molecular Biology of the Cell, Fourth Edition Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter 28/02/2002 1616 pages

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