Intercellular and intracellular signals

1. Intercellular and intracellular signals Primary signal molecules: small, apolar mol's, unstable compounds, small and large water-soluble molecules hormons, paracrine-, autocrine- and juxtacrine (contact dependent) factors growth-, death-, survival and differentiation factors Receptors:intracellular and cell surface receptorsreceptors with DNA binding, transcription regulating activity,G-protein coupled receptors, ion channel-linked receptors, enzyme-linked receptors Second messengers (intracellular signal molecules):cAMP, IP3, DAG, Ca ions, protein kinases/phosphatases, ubiquitin-ligases, proteases, (scaffold proteins, cytoskeletal structures, motor proteins) Targets:DNA - general transcription factor complexes, histon proteins (nucleosome), metabolic enzymes, RNA processing mechanisms (splicesomes), extracellular matrix

2. Growth-, survival- and differentiation factors On the basis of their biological activity we distinquish growth-, survival and differentiation factors Growth factors are mitogenic, elicit proliferation of the cell type,survival factors are needed for prolongation of life,differentiation factors trigger irreversible changes in morphology and metabolism (some of them are death-factors, as terminal differentiation – in certain cell types – leads to programmed death)death-factors, triggering programmed death, lysis or necrosis of cells These factors are pleiotropic: their effect depends on cell type, stage of differentiation, presence of other factors, etc. Survival factors can be growth factors for other cell types or cause apoptosis in another cell types. In general all these factors are referred to as growth factors (GF)

3. Receptor types Ion channel-linked receptors regulate the traffic or transport of specific ions. Ligand binding elicits opening of the channels. G-proteins coupled receptors activate G-protein trimers and indirectly (through the activity of G proteins) induce the synthesis of second messengers, like cAMP, IP3 and DAG. Ligand-binding induces oligo-merization of enzyme-linked (and signaling protein-linked) receptors, which leads to activation of different enzymes and second messengers.

4. Receptor types 1.Receptor tyrosine kinases A number of growth factor-, cytokine-, and some hormone receptors exhibit tyrosine-kinase activity Ligand-binding induces dimerization, activate the latent kinase, which results in the mutual phosphorylation of receptor subunits Second messenger molecules are attracted to the phosphotyrosine residues of the receptor The kinase also phosphorylates these, activating these second messengers platelet-derived growth factor PDGF receptor

5. Receptor types 1.Receptor tyrosine kinases A number of growth factor-, cytokine-, and some hormone receptors exhibit tyrosine-kinase activity Ligand-binding induces dimerization, activate the latent kinase, which results in the mutual phosphorylation of receptor subunits Second messenger molecules are attracted to the phosphotyrosine residues of the receptor The kinase also phosphorylates these, activating these second messengers In case of these receptors the subunit(s) of the receptor exhibit(s) kinase activity

6. Receptor types 2. Tyrosine kinase-linked receptors Other receptors are linked to kinases. The kinase is a separate molecule, which associates with the receptor after ligand-induced dimerization. The kinases phosphorylate each other, subunits of the receptor and second messenger molecules, attracted to the active receptor The receptor subunits have no kinase activity: receptor activation leads to kinase activation

7. Channel-linked receptors Activation of these receptors leads to the opening of specific ion channels These receptors can be localized in the plasmamembrane or in intracellular membranes (eg.: vitamin D3 receptor, IP3 receptor) ATP-gated receptors (P2X) playroles in contractility, tone, nociception, etc. are specific for calcium or sodium ions P2X vitamin D3 receptor

8. Channel-linked receptors(ligand-gated channels) A number of neurotransmitters have receptors with ion channel activity (cys loop type of receptors). These can be specific for Cl ions (eg. GABA receptors, histamine receptor) or for cations (nicotinic acetylcholin R, 5HT).

9. Channel-linked receptors(ligand-gated channels) NMDA (N-methyl-D-aspartic acid) receptor is one member of the glutamate receptor family. It has 4 transmembrane domains. Splice variants are present in different cell types. It is a Ca(Na)/K pump.

10. Mitogenic factors Each cell type have characteristic GF receptors. Only these can induce proliferation of the cell. Mitogenic factors are highly specific for cell type and degree of differentiation. The same factor might have other effects on other cell types (pleiotropic effect). (Tumor necrosis factor, TNF is a mitogenic factor for fibroblasts, but cytostatic on melanoma cells, inducesdifferentiation of myeloid cell, dedifferentiation in chondrocytes, inhibitsdiffferentiation in myoblasts and kills different tumor cell types, oligodendrocytes, endothelial cells Mitogenic factors: colony stimulating factors (CSF): for mieloid progenitors, interleukin-2 (IL-2): T-cells, IL-6: B-cells, erythropoetin: reticulocytes

11. Signaling pathways Receptors frequently induce several signaling paths, which can interfere, synergize or modulate each other. Some pathways are characteristic to certain cell types. Membrane-associated proteins are very important in signaling. Mitogenic factors activate themitogen-activated protein-kinase (MAPK)pathways These are parallel paths,serving different signals.All activate transcriptionfactors (eg. Ap1)

12. Signaling is an amplification cascade Each step amplifies the signal, more and more active molecules are generated Reversible phosphorylation is a key element of the signaling process Kinases phosphorylate tyrosine,serine, threonine amino-acids of proteins Activated proteins are either dephosphorylated by phosphatases or they are degraded (after ubiquinylation)

13. Signaling of receptor tyrosine kinases Mitogenic (growth) factors activate one of the mitogen-activated protein kinase pathways, each representing a chain of proteins with kinase activity.

14. Signaling of receptor tyrosine kinases The signal is amplified by each step. Activity of other enzymes are also modified by the kinases and other signal pathways might also be activated. The targets are DNA-binding transcription factors.

15. Mutations of the receptors or signaling molecules leads to disease Members of the FGF family of receptors from homo-and heterodimers with different specificities. Mutations affecting the ligand-binding domain might cause severe disease

16. Signaling of kinase-linked receptors Some growth factor receptors signal through the activation of protein kinases. Ligand binding activates receptors. The kinase is a separate molecule (JAK), which associates with the activated receptor (ligand-induced dimerization). Adapter molecules (eg. STAT) are attracted to the phosphorylated receptor and get activated (phosphorylated) by the kinase. Activated adapters dimerizeand are translocated into the nucleus. Their DNA binding modifies activity of many genes (they function astranscription factors),

17. Signaling of kinase-linked receptors

18. Signaling of kinase-linked immune receptors Ligand-specific and signaling subunits build up these receptors. The same signaling subunit can serve many receptors with different ligand specificity. This may cause competition of different ligands for the receptors.

19. Insulin receptor and signaling Activation of the insulin receptor triggers PI phosphorylation. An enzyme complex is assembled on the inner surface of the membrane. The activated kinases unlimately change the balance of glucose metabolism (in an opposite way what we learned aboutthe effect of glucagon)

21. Differentiation factors A differentiation factors – like mitogenic factors – highly specific for their target cells Receptors of differentiation factors is produced only in certain stages of differentiation and might disappear after one or several steps Differentiation factors are of different chemical nature: mostly proteins, but steroids and retinoids are also potent morphogens A typical differentiation factor, transforming growth factor (TGF) is a member of a superfamily of proteins (activins, inhibins, TGFs, bone morphogeic proteins, BMPs, etc.).Their structure, signaling pathways, functions remained highly preserved during evolution

22. TGF/TGF.R signaling Ligand binding activates the receptor (ligand-induced dimerization). Adapter molecules (SMADs) are attracted to the phosphorylated receptor and get activated (phosphorylated). TGF.R family members use specific Smad proteins for second messengers Smad1 to 3 and 5are signal trans-ducers, Smad4 is a co-Smad, while Smad6 and-7 are inhibitors of Smad signals The pathway is verysimilar to the STAT pathway

23. Survival factors and their receptors The population of certain cell types can be controlled at the level of proliferation or at the degree of survival: 1. if GF is not present, the cells are living in G1 or G0 phase (no DNA synthesis). Proliferation is triggered by the presence of GF. 2. GF is always present, the cells are continuously proliferating, however, their lifespan is limited (terminal dfferentiation leads to apoptosis) Apoptosis of these cells can only be prevented by survival factors Hemopoetic stem cells (HSC) require stem cells factor (SCF),interleukin-3 (IL-3), and flt-3 ligand (FL) for survival. Absence of these factors leads to death, not only growth-arrest. Survival factors:NGF (nerve GF): neurons, IGF-1 (insulin-like GF): many cell types, endothelin: endothel cells, BAFF: B-cells, leptin: eozinophil granulocytes, neurotrophins: neurons, embryonic stem cells, VEGF (vascular endothelial growth factors): capillary endothel, retina endothel Survival factor receptors belong to different receptor families (mostly GF.Rs)

24. Death receptors Certain cell types (eg. autoreaktív T and B cells) must be killedActivation of death receptors (fas, TNF.RI, NGF.R, TGF.R, etc.) leads to programmed cell death – in a cell type-specific way. The same receptors might function as growth factor receptors in other cell types death pathway survival pathway proliferation

25. Wnt signal: a special pathway Wnt signal also serves as a differeniation signal (ventral-dorsal) during ontogenesis The receptor is a 7TM domain GPCR, which activates more than one pathway In the absence of the ligand beta catenin is degraded. Activation of the receptor leadsto stabilization of catenin, allowing nucleartransport and gene regulation by activatingtranscription factor(s).

26. Catenin is a multifunctional protein beta catenin is associated with cytoskeletal structures linking cells of the same cell type together The Axin-APC-GSK complex (inducing phosphorylation-linked ubiquitination) degrades only free beta-catenin molecules, not interfering with the structural function of the protein

27. Wnt signal and differentiation Wnt can not activate its receptor in the posterior segment of the embryobecause of interference of other factors

28. The balance of kinases and phosphatases Signaling is influenced by the balance of kinases and phosphatases. Rafts (membrane microdomains) can influence this balance: while receptor and kinase are raft-associated, the phosphatase is not

29. The balance of kinases and phosphatases Signaling is influenced by the balance of kinases and phosphatases. This balance is cell type specific and can inhibit the generation of the signal Though the ligand inter-acts with its cognate receptor no signal is generated SHP-1 enzyme de-phosphorylates the subunits of the receptor and the second messenger molecules

30. Scaffold proteins Scaffold proteins are matchmakers,they catalyze the interaction of signal molecules Molecules participating in subsequent steps of the signal are bound to the surface of scaffold proteins, recognizing and modifying each other Heat shock proteins do the opposite:try to inhibit interaction of signaling molecules in the absence of ligand, preventing false signals

31. Pathways of TNF.R signaling Members of the TNF.R super-family can signal death as well as survival, proliferation, activation and differentiation. In different cell types different pathways dominate, other might be missing The pathways are under elaborate control mechanisms.

32. Differentiation steps In a cell at a certain stage of its differentiation - a set of transcription factors regulate the activity of genes, - a set of its genes show characteristic pattern of histone and DNA modification - the hnRNAs are spliced with characteristic splicing factors, resulting in a set of splice variant mRNAs, - the proteins are modified by characteristic processing enzymes - it results in a characterisitc enzyme activity pattern, metabolic activity and morphology - life span of the proteins is regulated by characteristic factors - the cell secretes specific proteins and exhibits a characterisitc set of receptors. The metabolic ativity and the morphology allows the cell to perform specific tasks, determines the way how to communicate with other cells and how to respond to external signals. Depending on the signals detected by its receptors, the cell can have different fates: proliferation, differentiation, activation or death

33. Differentiation steps The differentiation factor interacting with the receptor of the cells induces a signal process resulting in specific changes: - in the activity of enzymes (different phosphorylation patterns, different activities), - in the composition of transcription factors, - in the modification of histones and DNA, - inducing synthesis of a new sets of hnRNAs, - splicing of these hnRNAs with a new set of splicing factors, - resulting in the synthesis of new splice variant proteins and new sets of enzymes, receptors, ECM proteins, - changing the pattern of protein-modifying and protein-degrading enzymes, - resulting in different metabolism,. The new metabolic activity might mean changed morphology and new functions. The cell starts to produce new receptors and signal molecules, different ECM: its communication with other cells has also changed. In many case the differentiation process is irreversible.

34. Signal and co-signal In cell-fate decisions signals frequently need a corroborating co-signal to be effective In other cases, the presence of a co-signal alters the meaning of the original signal In immune cells: antigen + co-signal: survival, activation, proliferation antigen alone: apoptosis or anergy

35. Juxtacrine signal and ECM signal In cell differentiation ligands originating from neighboring cells (cell adhesion molecules, juxtacrine factors) or the extracellular matrix (ECM) play very important roles The signal is usually bi-directional. Both cells differentiate (in the same or different directions)

36. ECM-triggered signaling

37. ECM-triggered signaling The cells produce cell-type specific ECM molecules. These proteins activate different signaling pathways in the signaling and the neighboring cells. These signals harmonize cells of a certain tissue, create links between different cell types or induce differentiation of cells. Similar cells are linkedtogether by homofil-,different cell types by heterofil cell adhesionmolecules (CAMs)

38. Cross-talk of signaling pathways • Cells are exposed to simultaneous signals. The signaling pathways and signaling molecules modulate each other: they synergize, interfere or modify each other. • The effect of a certain factor depends on the presence of all other factors

39. The EGF.R signaling pathways

40. Alternative pathways result in different effects

41. Survival factors Apoptosis-prone cells survive only in the presence of survival factors Neurons: NGF, neurotrophic factors, hemopoetic SC: SCF, IL-3, flt3-L The survivalfactors block thepro-apoptoticsignal cascades,protecting the life of the cells