Communication between cells, signal molecules

1. Communication between cells, signal molecules • Hormones, growth factors, cytokines • Their receptors • Intracellular signaling events

2. Communication between cells, signal molecules Communication between neighboring cells desmosomes, nanotubules (coordination of metabolism)juxtacrine signaling Long distance communication between cellshumoral connection (endocrine, paracrine) Physical connection between cells (neuronal)part of this is also humoral: neurotransmitter molecules act as paracrine signals Humoral communication Unity of the organism is based on coordinated communication between cells The cells continuously exchange signals among each other The signal molecules are hormones, survival-, growth- and differentiation factors, cytokines and chemokines. Different cell types produce and detect different signals (Communication within individuals of one species: pheromones, among different species: odors, scents)

3. Cell-cell communication: no signal molecule Communication between neighboring cells: no signal molecule is needed, direct link (desmosomes, tunneling nanotubules) between the cytoplasms (metabolism of the cells is coordinated) Change in intracytoplasmic Ca++ ion concentration spreads from one cell to other in case of desmosomally linked cells

4. 15 mm Tunneling nanotubules (TNT) link cultured myeloid THP-1 cells

5. Spread of Ca++ ion flux (change in concentration) in TNT-linked, Fura-2 lebeled cells

6. Spread of Ca++ flux in TNT-linked dendritic cells (after stimulation by micropipette tip)

7. Spread of Ca++ ion flux in cultured THP-1 cells

8. Definitions The molecules, produced by the signaling cell (hormones, cytokines, growth factors, chemoattractants, neurotransmitters, etc.) are usually called ligands. The molecules that detect them and interact with them with high specificity and high affinity are called receptors. Receptors are located in the cytoplasm or on the plasmamembrane. The receptors we discuss today are either transcription factors or able to activate transcription factors by intracellular signaling mechanisms.

9. SignalingApprox. half (40-55 %) of our proteins/genes with known functions participate in signaling events:ligands, receptors,second messengers,kinases, regulators,ion channels, etc.

10. Cell-cell communication: signal molecules Humoral communication between cells: endocrine and paracrine contact Membrane penetrating (small, hydrophobic)molecules, cytoplasmic receptors, direct effect on gene activity Hydrophilic molecules,cell-surface receptors, intracellular signaling second messengers sophisticated signaling and signal-amplification mechanisms

11. Hormones Endocrine signaling: hormons are produced by endocrine glands, the extracellular signal is secreted into the blood stream, their effect is very specific Paracrine signaling molecules might circulate outside the blood vessels Autocrine factors stimulate the producing cells: in case of mitogenic factors it could lead to unlimited proliferation Signaling: 6-7 steps production of the signal, export (secretion), transport, detection (receptor), intracellular signal- transduction,metabolic response,termination of the signal: decomposition of the molecule(s) participating in the signal

12. Classification of signal molecules Cell biologists, physiologists, immunologists use different terms - Insulin is regarded as a hormone (secreted by endocrine beta cells) or as a growth factor (induces proliferation of cell types) - Interleukin-2 is a regulatory cytokine, also a T-cell growth factor - Erythropoetin is a differentiation factor, a cytokine and a hemopoetic hormone The same moleculemight have many different functions (pleiotropy) and different names

13. Hormones 1. Hydrophobic hormones with intracellular receptor retinoids (vitamin A-related compounds), steroid hormones, tyroxine – very hydrophobic molecules, in the circulation they form complexes with carrier proteins they can cross the plasmamembrane toget into the cytoplasm their cytoplasmic receptors are DNA-binding transcription factors,regulating gene activity Retinoids are important morphogenes, playroles in differentiation,cell prolif. and death,photosensitivity, - used in the clinic (anti-leukemic agent)

14. Hormones 1. Hydrophobic hormones with intracellular receptor retinoids, steroid hormones, tyroxine – very hydrophobic molecules, in the circulation they form complexes with carrier proteins they can cross the plasmamembrane toget into the cytoplasm steroids regulate mineral metabolism, sexual dimorphisms and sexual functions,tolerance of stress, andimmune responses Animal and plant steroids,natural and synthetic steroids in the clinic

15. Cytoplasmic receptors The ligands (steroids, retinoids, tyroxin) circulate in pockets of carrier proteins Dissociation of the complex results in free ligands, that penetrate the cell cytoplasm The receptors exhibit highaffinity and high selectivity The receptor-ligand complexbinds DNA in a sequence-specific manner after nuclear translocation Binding to individualenhancer elementsregulates activity of genes thyroxin

16. Working mechanism of cytoplasmic receptors Monomers of receptors form complexes with chaperone molecules. These complexes interact with ligands. The ligand-induced change in receptor conformation destabilizes the complex: The receptor forms dimers,translocates andrecognizes the properDNA sequences,binds to them andinteracts with other transcription factorsmodifying their activity

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20. Hormones and other ligands 2. Lipophil molecules with cell surface receptors eicosanoids: leukotrienes, prostaglandins (16 species, PGA – PGI), prostacyclines, tromboxanes they are formed from the fatty acid chain (arachidonic acid) of phospholipids, they have very limited life span: short distancesignal molecules

21. Hormones and other ligands 2. Lipophil molecules with cell surface receptors eicosanoids: leukotrienes, prostaglandins (16 species, PGA – PGI), prostacyclines, tromboxanes they are formed from the fatty acid chain (arachidonic acid) of phospholipids, they have very limited life span: short distancesignal molecules the have roles in blood clotting, wound healing, inflammation,muscle contraction (smooth muscle cells of capillaries, uterine contraction, etc.

22. Hormones and other ligands 3. Small, hydrophilic molecules with cell surface receptors histamine, epinephrine, dopamine, serotonin, acetylcholin, stb. Serotonin

23. Seven transmembrane domain (7TD) receptors Receptor of glucagon belongs to the superfamily of seven transmembrane domain receptors. This is the largest receptor protein family: light-, scent-, taste- and chemoattractant-sensing, pain-modifying, immune- and neuronal receptors, receptors influencing blood clotting,muscle function, etc. belong to this group of proteins.

24. G-protein linked/coupled receptors These receptors signal through trimeric G-proteins. These G-proteins are GTP degrading enzymes (GTP-ases), using the energy of GTP to activate other enzymes. Activation of the receptor by the ligand leads to the activation of G-protein, binding GTP (subsequently GTP is cleaved to GDP and the protein returns to inactive form). G-proteins regulate the activity of other enzymes – depending on the type of receptor – they can activate of inhibit. One G-protein can influence the activity of many enzyme molecules during its short active period.

25. Working mechanism of G-proteins

26. G-protein-linked (7TD) receptors

27. G-protein-linked receptors, adenylate-cyclase Ligand-binding activates the system. Receptor-induced activation of G-proteins leads to the dissociation of the trimeric G-protein and activation (inhibition) of other enzymes, eg. adenyl-cyclase Adenyl-cyclase convertsATP into cyclic AMP, a second messenger.

28. Adenylate cyclase and cAMP

29. Signaling is an amplification mechanism Steps of signaling form a signal- amplification cascade cAMP molecules liberate the active protein kinase-A from an inhibitory complex cAMP can directly influence the activity of DNA-binding proteins, modifying activity of genes

30. GPLR and the Ca ion signal G-proteins can activate phospholipase-C. PLC liberates phosphorylated inositol (IP3) from phosphatidyl-inositol (PI), leaving diacyl-glycerol (DG). Both molecules are second messengers. Acting on Ca-channels and Ca pumps IP3 releases Ca ions from cytoplasmic Ca stores (ER)and allow extracellular Cainto the cells. Ca ions modifythe activity of many differentenzymes. The metabolic ac-tivity of the cell has changed

31. Ca signalThe level of Ca is very low in the cytoplasm. Ca is stored in vesicles, released by the signal.

32. Regulatory role of cAMP cAMP has regulary roles at different levels: indirectly influencing enzyme activity by releasing Ca ionsdirect regulation of enzyme activity andcontroling gene activity by modification of transcription factors

33. Regulatory role of the Ca signal The same ligand induces different metabolic changes in different tissues (cell types). It is called pleiotropic effect. Signaling components, enzymes, transcription factors are different in different cell types. The same signal (eg. Ca flux) leads to very diverse metabolic changes. Acetylcholine, a neurotransmitter generates many types of responses.

34. The life span of the signal has to be limited The signal results in changes – no need to prolong the signal. Cells should be able to respond to signals again. Signals must have an end. How to terminate signaling? Production and life-time of signal molecules are regulated Receptors (ligands) can be modified, degraded or downregulated Second messengers have short lifespan Signal-elicited modifications are reversible

35. Summary Hydrophobic ligands have cytoplasmic receptorsthese receptors have direct, gene regulatoryeffect, binding to control sequence elements Chaperones participate in the signal process Hydrophilic ligands have cell surface receptors. These receptors use sophisticated intracellular signaling pathways to regulate gene activity and metabolic processes. There are many types of cell surface receptors. G-protein-linked (seven transmembrane domain) receptors are one, important type of these receptors.

36. endocrine, paracrine, autocrine, juxtacrine factors, ligands and receptors hormones, survival-, growth- and differentiation factors, cytokines and chemokines extra- and intracellular signaling lipophilic and hydrophilic hormones/ligands steroids (effects of steroids) retinoids as morphogens and anti-leukemic drugs thyroxine intracellular receptors of cell penetrating ligands role of chaperones in signaling processes intracellular receptors as DNA-binding proteins intracellular receptors are transcription factors cell surface receptors lipophil molecules with cell surface receptors eicosanoids, tromboxanes, prostanoids receptors for light, smell and tastes seven transmembrane domain receptors = G-protein coupled/linked receptors G-proteins adenylate cyclse, cAMP phospholipases, PLC phosphatidyl inositol, PIP2, IP3, DAG protein kinases, PKA enzyme regulation by phosphorylation intracytoplasmic Ca stores Ca mobilization, Ca flux limiting signal duration receptor endocytosis/downregulation reversible modification of signal molecules You should remember: