Mechanisms of cell communication part I & II

1. Cell biology 2014 (revised 29/1 -14) Lecture 4 & 5: Mechanisms of cell communication part I & II Cell Biologyinteractive media  ”video” or ”interactive”

2. Differentiate Proliferate Secrete Die Move x x Cell communication Signal molecules/proteins All diseases involve changes of normal cells. In some cases, these changes may affect other cells of the individual

3. 1. Target cell Target cell response 5. 2. 1. 2. 3. 4. 6. 6. Termination of signal 5. 3. 4. Events during cell communication Regulated synthesis…..….. Producer cell .. or regulated release of a signaling molecule (Transport of signaling molecule to target cells) Binding of the signaling molecule to a specific receptor on/in a target cell Activation of a transduction chain “hormone" = to urge on/impulse

4. Signaling receptor diversity The mammalian genome encodes for thousands of signaling receptors - Many of these are targets for drugs Tissue specific expression: Each individual animal cell express only some of these receptors

5. Membrane permeability • Hydrophobic molecules O O O O O N N Cholesterol Cortisol Testosterone • “Large” uncharged polar molecules C C Glucose • Charged • molecules Cl- Na+ Ions Amino acids

6. Localization of signaling receptors Receptor in cytosol Receptor in nucleus Hydrophobic molecules Hydrophilic molecule (and proteins) Receptor on plasma membrane Other compounds than the natural ligand may interact with a receptor – some are used as drugs (legal & illegal) “natural ligand” = an endogenous receptor binding molecule hydrophobic lipohilic non-polar (often used as synonyms)

7. Receptor agonists and antagonists Other compounds than the natural ligand may bind a receptor Agonists: mimic completely, or partially, the action of the endogenous ligand Antagonists: bind to receptor without activating it  block the action of the “natural” ligand Adrenalin Phenylephrine (natural) (selective agonist) One of the action of adrenalin is to cause a dry mouth in the fight-or-flight reflex. Phenylephrine is used in many “cold-relief” drugs to prevent excessive nasal mucous secretion

8. A Signaling by secreted ligands: Autocrine Paracrine C B Endocrine D Neuronal/synaptic E Five modes of cell communication Surface receptor Intra-cellular receptors C B D Bloodstream A E Contact dependent signaling: Ligands on the cell surface Neuron "crinis" = secrete

9. A Contact-dependent signaling Target cell Signaling cell Receptor/ligand • Contact-dependent signaling uses ligands and receptors that are plasma membrane-bound: • Persistent signals (uni- or bidirectional) • Directed toward neighboring cells

10. A Distinct types of contact-dependent cell signaling Cell surface receptors that mediate cell-to-cell adhesion (cadherins) and cell-to-ECM interaction (integrins) are also involved in signaling. Importantfor: Development Growth control Survival Gap Junctions permit free passage of small molecules between adjacent cells Important for e.g., synchronous heart contraction Cadherin Gap junction Integrin

11. B Paracrine signaling Signaling cell Adjacent target cells Paracrine signaling involves secretion of a ligand that act locally on cells with the appropriate receptors: Local effect  "para" = near

12. Autocrine signaling implies that a cell secretes a ligand that it responds to itself Signaling and target cell C Autocrine signaling "autos" = self ?

13. D Endocrine signaling Endocrine cell secreting Distant target cells Endocrine signaling involves a signal molecule (poly-peptide or steroid hormone) produced by an endocrine cell. "endo" = inside/within "crinis" = secrete Each endocrine cell secrete only one type of signal molecule! The hormone travels through the blood system: Global signaling with long-term effect Relatively slow responses - the signaling molecule have to travel through the blood systems before reaching a target cell

14. E Neuronal/synaptic signaling Target cell Signaling cell Release of neurotransmittor Axon Cell body of a neuron Synapse "syn" = together "haptein" = hold onto Neuronal/synaptic signaling is mediated by neurotransmitters released at the interface between the signaling and the target cell, called synapse. The release of neurotransmitters at the synapse is controlled from the cell body through electrical signals. Neurotransmitters bind cell surface receptors. - Acts rapidly and transiently on the target cells

15. Neuroendocrine integration • Hormone secreting glands in the brain link neuronal signals and peripheral endocrine glands. • Fight-or-flight reflex: the Hypothalamic- Pituitary-Adrenal (HPA) system • The adrenal gland responds to both the hormone (ACTH) and a nerve signal • ACTH Adrenal Cortex cortisol • Increased blood levels of lipids etc. etc • Nerve signaladrenal medulla adrenaline •  Increased blood levels of lipids & glucose • etc. etc. • Endocrine cell: a cell within an endocrine • gland that release a hormone into the circulating blood in response to a neural (synaptic) or hormonal stimulus *Gland *Gland kidney

16. Signaling molecules Signaling molecules are chemically diverse: - Gases: nitric oxide, carbon monoxide - Steroids: testosterone, cortisol, etc. - Proteins: insulin, glucagon, etc. - Amines: catecholamines, acetylcholine “Ryss 5a”: A mix of synthetic anabolic steroids ( muscle growth) Membrane permeable Membrane impermeable Molecules typically produced and released by one cell and recognized by another cell

17. Fast versus slow signal transduction events Signal Altered gene expression Altered protein function DNA mRNA Fast (<seconds) mRNA Altered protein level An altered cytoplasmic signaling protein Protein Slow (minutes to hours) Cell response

18. CH2 CH2 CH2 O O O NO2 NO2 NO2 Signaling with nitric oxide gas • Nitric oxide (NO) acts as a paracrine signal, only affecting local • area, due to its short t1/2 (1-5 seconds) • Produced by nitric oxide synthase through the deamination • of the amino acid arginine • Nitric oxide is a very potent vasodilator (blood vessel dilatation) Nitroglycerin is converted in blood to NO (used to treat coronary artery disease since 1878)

19. Three types of cells dedicated to contraction • Skeletal muscle • Cardiac muscle • Smooth muscle cells: • i) surrounds hollow organs – intestines and blood vessels • ii) arrectorpili muscles attached to hair follicles All three muscle cell types contains filaments consisting of actin and myosin, which may contract and slide apart

20. Vasodilatation through nitric oxide signaling Neuron Acetylcholine Blood vessel Endothelial cell Arginine NO (Nitric oxide) Diffusion to adjacent smooth muscle cell Smooth muscle cell ”2nd messengers” Relaxation of smooth muscle cell Increased blood flow

21. Cytosolic signal mediators: second messengers 1st messenger: the external signaling molecule (e.g. Nitric oxide) 2nd messenger: the molecule that transfer the signal in the cytosol cAMP, cGMP and Ca2+ are the classical 2nd messengers =1 mM Ca2+ Ca2+ Adenylyl cyclase Ca2+ =10 nM Ca2+ Guanylyl cyclase Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Video 15.1-calcium_signaling

22. P P P P P P P Effect of nitric oxide on smooth muscle cells Guanylyl cyclase Nitric oxide GTP Cyclic-GMP phosphodiesterase (constitutively active) + Cyclic GMP Activation of an “in-ward” Ca2+-pump in membranes of intra-cellular Ca2+-stores Viagraä GMP Relaxation of smooth muscle cells and increased blood flow Low [Ca2+] makes contractile filaments (actin and myosin) slide apart

23. Receptor translocation into the nucleus  specific transcription 3. 2. 1. 2. 3. Signaling by intracellular receptors – part I Hydrophobic ligand (e.g. Cortisol) Plasma membrane 1. Cortisol diffuse through the plasma membrane NLS Combined receptor/ transcription factor NLS Binding displaces a protein that masks an NLS on the cortisol receptor = DNA NLS Target genes

24. Inhibitor 1. 3. 2. 3. 2. 1. Signaling by intracellular receptors – part II Hydrophobic ligand Plasma membrane Inhibitor Target genes Target genes The DNA-binding receptor/transcription factor is inactive The ligand (e.g. sex hormones) diffuses into the nucleus The ligand displaces the inhibitor

25. 1. 3. 2. General principle of cell surface receptor signaling Signal molecule (Ligand) Reception P. M. Receptor Cytosol Signal transduction cascade comprising: i. molecular switches ii. 2nd messengers Gene regulatory protein Metabolic enzyme Etc. Response

26. OH I. Molecular switches in signal transduction A signal that can be switched on, also needs to be switched off (all signals are more or less transient) 1. Protein phosphorylation The most common ‘on-off’ switch is provided by protein phosphorylation - O O P - + ATP Kinase O O Serine, threonine or tyrosine Serine, threonine or tyrosine Phosphatase Kinase : ~1000 protein kinase genes in vertebrates. Some have only a single substrate. Others are “multi-functional” and may have >10 substrates

27. II. Molecular switches in signal transduction 2. GTP binding proteins (G-proteins) Another ‘on-off’ switch is provided by regulatable GTP-binding and hydrolysis GDP GTP >> GDP GTP Guanine-nucleotide Exchange Factor (GEF) GDP GTP Inactive Active GTPase Activating Protein (GAP) P Molecular_models 15.5-Ras (one PO4 makes the diff.)

28. P P P Signal transduction cascades A single cell surface receptor may activate several signal transduction pathways This involves various G-proteins, 2nd messengers and protein kinases Protein kinases at the end of a cascade may have many substrates P. M. cGMP cAMP Ca2+ GTP Kinase Response: Gene regulation Metabolism Etc.

29. Three main classes of cell-surface receptors G-protein coupled receptors Receptors with intrinsic enzymatic activity Ion channel coupled receptors Ion Ion ZZZ ZZZ ZZZ Ligand Ion

30. G-protein coupled receptors (GPCR) A hallmark of GPCR´s is 7 trans-membrane spanning regions ZZZ 3. G-proteins may regulate enzymes or ion channels 1. Ligand binding conformational change 2. A specific G-protein is recruited and activated G

31. 3. 4. 2. 1. Down-stream effectors of various G-proteins ATP GTP Guanylyl cyclase Adenylyl cyclase Cyclic AMP Cyclic GMP Ion channels Ion Phospholipase C Ion Increase in cytosolic and activation of protein kinase C Ca2+ Ion

32. g I. Regulation of hetero-trimeric G-proteins =GEF GDP GTP Complex dissociate upon GTP binding >> GDP GTP RGS GDP GTP b + Inactive Active a b a g =GAP a-subunit and/or b,g-subunit can activate or suppress different downstream targets P RGS: Regulator of G-protein Signaling

33. g II. Regulation of hetero-trimeric G-proteins No ligand (default state) P.M. GDP a b g Ligand binding causes a conformational change P.M. GTP GDP b a a + b g GTP GDP The G-protein is recruited to the receptor, which acts as a GEF  the a-subunit exchanges GDP for GTP  dissociation of an active a-subunit

34. g Phospholipase C-b (PLC-b) Adenylyl cyclase III. Regulation of hetero-trimeric G-proteins The intrinsic GTP hydrolysis is slow but RGS, an a-subunit specific GAP, catalyzes hydrolysis. This terminates the signal P.M. GDP GDP GTP b a a a + b g RGS P A family of -subunits with distinct functions GTP GTP GTP as aq ai Anim. 15.3-G-protein_signaling Alberts et al: Table 15-3 (tissue specificity)

35. P P P P P P P Adenylyl cyclase activation by the as-subunit of G-proteins Adenylyl cyclase P.M. GTP as Caffeine ATP Cyclic-AMP phosphodiesterase (constitutively active) + Cyclic AMP AMP

36. P P 2. 1. 2. 1. Cyclic AMP second messenger signaling Cyclic AMP Cyclic AMP activates Protein kinase A (PKA), which can regulate: Metabolism Gene transcription Inactive PKA CREB Active PKA Glycogen phosporylase Glycogen phosporylase CREB Target genes Glucose-1- phosphate Glycogen

37. P P P P Summary of the cyclic AMP signaling cascade GTP GEF (GPCR) as CREB Regulates transcription -Regulated DNA binding Cyclic AMP PKA Regulates metabolism Adenylyl cyclase ATP • Glycogen: • Stored in muscles and liver • Rapidly available energy source • Work/stress  adrenalin  cAMP  PKA    Glycogen breakdown Glycogenbreakdown Alberts et al: Table 15-1 (tissuespecificresponse) Anim. 15.4-cAMP_signaling

38. Signal induced cleavage of phospholipids External signals may activate distinct phospholipases that cleave phospholipids at specific sites and thereby catalyze the formation of various molecules with signaling properties Precursors for various signaling substances Fatty acid Fatty acid Phospholipase A2 Phospholipase A1 Glycerol Phospholipase C Phosphate Soluble compounds  release into the cytosol Phospholipase D Variable

39. OH P P P P P P 1. 2. 1. 2. Phospholipase C activation generates two 2nd messengers Phosphatidylinositol 4,5- bisphosphate, PI (4,5)P2 Diacylglycerol (DAG) Inner leaflet of plasma membrane Fatty acid Fatty acid Fatty acid Fatty acid GTP aq Phospho- lipase C-b (PLC-b) Glycerol Glycerol aq-subunit activates PLC PLC cleaves PIP2, generating the two 2nd messengers DAG and IP3 Inositol 1,4,5- triphosphate, IP3

40. P P P 4. 1. 3. 2. 2. 3. 4. 1. Role of the 2nd messengers IP3 and DAG Inner leaflet of plasma membrane DAG DAG recruits PKC to plasma membrane OH Calmodulin PKC PKC Ca2+ Ca2+ IP3 mediate release of Ca2+ from ER Ca2+ Ca2+ Calmodulin Ca2+ Ca2+ DAG and Ca2+ activates PKC Ca2+ Ca2+ Ca2+ activates calmodulin to terminate signal by pumping Ca2+ back into ER IP3 Calmodulin regulated Ca2+ pump in ER Ca2+ Ca2+ IP3 regulated Ca2+ channel

41. Inhibitory P P Catalytic Resting state Inactive Increased cytosolic Ca2+ Ca2+ Ca2+ Ca2+ Calmodulin Calmodulin Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+/calmodulin dependent protein kinase (CaMK) Dephosphorylation Partially active Ca2+ Ca2+ Calmodulin Calmodulin Ca2+ Ca2+ Autophosphorylation Activated Fully active Molecular_models 15.6-calmodulin

42. P P P P P P Other regulated enzymes Ca2+ Summary of G-protein signaling throughPLC-β Both PKC and CaMK have many potential (tissue specific) substrates GTP GEF (GPCR) aq PKC PLC-b DAG OH CaMK Ca2+ Ca2+ Calmodulin Ca2+ Ca2+ Ca2+ IP3 STOP Etc! Termination of signal Ca2+

43. P P Enzyme linked receptors Many variants on this theme – here we focus on: Receptor tyrosine kinases Receptor serine/threonine kinases Single pass transmembrane receptors. Ligand binding cause dimer formation and consequent “auto”-phosphorylation Tyr Ser/Thr Hetero-dimers Homo-dimers Alberts et al: Table 15-4 (tissuespecificRTK’s) Jenkinson : RTK - dimerization

44. P P P P P P Signaling through Receptor Tyrosine Kinases Single pass transmembrane protein Ligand binding causes receptor dimerization P. M. Kinase domain Kinase domain Kinase domain Kinase domain Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr Inactive receptor monomers Active receptor dimer Trans-phosphorylation of tyrosine residues Cis- prefix means "on this side" Trans- prefix means "across"

45. P P P P P P P Tyr Tyr SH2-proteins binds at specific phospho-tyrosines Regions containing phospho-Tyr may serve as specific docking sites for SH2 domain-containing signaling proteins (SH = SrcHomologydomain) Phosphatidyl- inositol (PI) Monomeric G-protein P. M. GDP GTP Ras Ras 3 Kinase domain Kinase domain Ras GEF (Sos) Tyr Tyr PI-3 Kinase SH2 SH3 Tyr Tyr These can be enzymes…. ………….or they act as adaptors for signaling proteins Fig. 15-55

46. P P P P P 2. 1. 1. 2. Phosphorylation cascade downstream of Ras P. M. GTP Ras Raf Altered protein function Altered gene expression Mek Mek Erk (MAPK) Erk Erk Cytosolic target proteins Target genes

47. P Dephosphorylation 1. 3. 2. Termination of RTK/Ras/MAPK pathway Receptor and ligand internalization Ras GTP hydrolysis GDP GTP Ras Ras Ras GAP Fusion with endosome Note: Signaling receptors are rarely recycled Erk (MAPK) Fusion with primary lysosome  degradation Erk Phosphatase Anim. 13.3-receptor_endocytosis (Note: vesicle fusion with endosome)

48. P P P P P P Fatty acid Fatty acid Glycerol Phosphate Inositol I. PI-kinases act at specific positions of the inositol ring Extracellular space Cytosol Phosphatidylinositol (PI) PI – phosphorylation cycles on inositol ring position 4 & 5 5 4 PI kinase PIP kinase 3 PI(4,5)P2 PI(4)P Inositol

49. P P P P P P P P 1. 2. 2. 3. 3. 1. II. PI-3 kinase completes a PH-domain binding site Phosphatidyl- inositol (PI) PI(4,5)P2 PI(3,4,5)P3 PTEN PI-3 Kinase 3 3 PI-3 kinase Activated receptor recruits and activates PI-3 kinase PI-3 kinase phosphorylates PI(4,5)P2 to generate PI(3,4,5)P3, which will serve as a docking-site for a family of signaling proteins with a “PH-domain” (PH= Pleckstrin Homology) PTEN removes phosphorylation on position 3 on PI(3,4,5)P3 to terminate signal

50. P P P P P P P P P P P P P P P P P P PKB/Akt PI(3,4,5)P3 brings PDK1 and PKB/Akt into proximity through their PH-domains PKB/Akt PDK1 2. 1. 2. 1. III. PKB/Akt activation downstream of PI-3 kinase 3 3 3 3 PKB/Akt PDK1 PH-domains PDK1 phosphorylates PKB/Akt thereby mediating its activation