MUNI SCI Cell signaling RNDr. Jan Škoda, Ph.D. Department of Experimental Biology Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Outline - Principles of cell-cell communication - Signals - their origin and distance of action - Intracellular signaling pathways -Chemical nature of signals - Examples of signals and their effects in the cell 2 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Communication - Response to the presence of other cells - influence one another's behavior in accordance with the changes in their environment - Prokaryotes: quorum sensing - population density coordinate motility, antibiotic production, spore formation, conjugation -Yeast: mating factor - peptide, signal for mating - Multicellular organisms: homeostasis, development, coordination of activities in response to the environment (food, photoperiodism, circadian rhythms, predators...) 3 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) fl Principles of cell communication 4 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Principles of cell communication 1- Signaling molecule (a ligand) produced by the signaling cell 2. Signaling molecule binds to a receptor at the target cell (at the membrane or internal) 3. Activated receptor induces internal signaling pathways leading to effectors = signal transduction (conversion of an extracellular molecular signal into a different form of messengers) 4. Effectors control the cell response EXTRACELLULAR SIGNAL MOLECULE RECEPTOR PROTEIN plasma membrane of target cell INTRACELLULAR SIGNALING PROTEIN EFFECTOR PROTEINS metabolic gene regulatory cytoskeletal enzyme protein protein I I I altered metabolism altered gene expression altered cell shape or movement 5 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Signal production Plasma a Membrane ^ - Utilizes vesicular transport and exocytosis (=^t^\±J^ -Signaling molecules secreted or presented at the cell surface - Different distance of activity - Contact, local, distant CONTACT-DEPENDENT PARACRINE SYNAPTIC ENDOCRINE Direct communication - Gap junctions in animal cells - Plasmodesmata in plant cells -Channels allowing exchange of ions and small soluble molecules (not macromolecules) Plasma membranes Gap junctions Plasmodesmata between animal cells between plant cells - Bidirectional signaling, follows concentration gradient - Signal molecules can spread to cells that would not otherwise receive it (synchronization of activities) 7 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Contact-dependent signaling Juxtacrine signaling Signaling molecule immobilized in the membrane of the signaling cell Contact with the receptor of the target cell Mediates contact of cells Important in development and immune responses ■ Notch, Programmed death-ligand 1, ephrin signaling CONTACT-DEPENDENT signaling cell target cell membrane-bound signal molecule 8 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Paracrine signaling Secreted ligands, local mediators ■ Signaling molecules diffuse locally in the extracellular space Signals activate receptors of neighboring cells E.g., regulation of inflammation or proliferation during wound healing vs. autocrine signaling: "self-activation"; triggers response in the producing cell; common in cancer cells PARACRINE ft? Target sites on same cell 9 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Synaptic signaling - Neurons transmit electrical impulses along axons - Long distances, fast, dedicated structures - Synapses - sites of cell-cell communication - Release of neurotransmitters - electrical signal converted into chemical 10 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) SYNAPTIC neuron axon cell neurotransmitter body target cell Endocrine signaling Long distance signaling, whole organism Slower but longer-acting Via bloodstream in animals, phloem in plants Systemic coordination of activities (different responses in different cells) ENDOCRINE endocrine cell receptor target bloodstream target cell 11 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Receptor activation and cell responses Receptors are activated by specific ligands Receptors bind ligands with high affinity - ligands can act at very low concentrations Expression (presence, concentration) of the receptor dictates sensitivity of the cell to the signal Cell surface and intracellular receptors ►Action CELL-SURFACE RECEPTORS plasma membrane cell-surface receptor protein hydrophilic signal molecule Receptors & ligands: "Lock & key" mechanism INTRACELLULAR RECEPTORS small hydrophobic /signal molecule V carrier protein target cell target cell nucleus intracellular receptor protein 12 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Not all ligands activate the receptor Endogenous agonists: growth factors hormones, neurotransmitters Agonists: chemicals binding and activating the receptor Antagonists: chemicals blocking the receptor - might have higher affinity than endogenous agonists = concentration and affinity-dependent effects HOmONE or receptor neurotransmitter kfcctrtok agonist antagonist 13 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Receptor activation and cell responses - Downstream signaling pathways induce effectors and context-dependent response Major effectors: -Gene regulatory proteins - Enzymes - Cytoskeletal proteins Transcription, metabolism, movement, differentiation, cell death... ▲ j EXTRACELLULAR SIGNAL MOLECULE RECEPTOR PROTEIN plasma membrane of target cell INTRACELLULAR SIGNALING PROTEINS X •—• metabolic gene regulatory cytoskeletal enzyme J protein J protein 1 altered altered gene altered cell metabolism expression shape or movement EFFECTOR PROTEINS SECRETORY CELL — Hormone molecule VIA BLOOD Signal receptor^ •a VIA BLOOD SECRETORY CELL Q J—Hormone // molecule Signal transduction pathway r / TARGET CELL I OR Cytoplasmic response Nuclear response NUCLEUS (a) Receptor in plasma membrane TARGET CELL Signal — receptor Signal transduction and response - Synthesis of specific proteins T. j mRNA I NUCLEUS (b) Receptor in cell nucleus 14 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Cell responds to a spectrum of signaling molecules Different sets of receptors at different cells Combination of signals shape the final response SURVIVE i GROW + DIVIDE I s t \ s i \ DIFFERENTIATE DIE i» l/. I—apoptotic + cell 15 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) One signal triggers different responses -Acetylcholine - Inhibitory effects in heart muscle cells ■ Stimulation of skeletal muscle cells - Stimulation of salivary gland cells - Differences in receptors - Some ligands bind to more receptors - Differences downstream of the receptor - signal processing ■ Components of the signaling pathways, expression profiles and effector proteins (A) acetylcholine O CH, II 1/ H3C —C —O —CH2—CH2 —N+-CH3 CH3 (C) skeletal muscle cell CONTRACTION (B) heart muscle cell receptor |ww\ protein MM acetylcholine DECREASED RATE AND FORCE OF CONTRACTIOI N (D) salivary gland cell ECRETION 16 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Ligands binding to intracellular targets - Diffusing gaseous and hydrophobic signaling molecules Nitric oxide - From arginine - NO synthases - Binds to guanylyl cyclase -> cyclic GMP -> cGMP activates effectors - Fast response - Acts locally (+ H20, 02 ^ nitrates, nitrites) - cGMP breaks down by phosphodiesterase I activated nerve terminal -acetylcholine V NO bound to activated NO synthase (NOS) guany|y| cyclase RAPID DIFFUSION OF NO ACROSS MEMBRANES endothelial cell 5!cJIeS RAPID RELAXATION OF SMOOTH MUSCLE CELL smooth muscle cell 17 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Nitroglycerin Viagra (Sildenafil) -Treatment of angina episodes - Converted to NO -> relaxed blood vessels -Inhibits cGMP phosphodiesterase in the pen -> prolonged activity of cGMP = relaxed blood vessels 18 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Ligands binding to intracellular targets Diffusing gaseous and hydrophobic signaling molecules Cortisol Steroid and thyroid hormones - Bind to intracellular nuclear receptors - Nuclear receptors contain DNA-binding domain -Active nuclear receptors can bind to DNA and regulate transcription plasma membrane conformational nuclear change activates receptor receptor protein protein CYTOSOL NUCLEUS activated receptor-cortisol complex moves into nucleus activated j z: target gene activated receptor-cortisol complex binds to regulatory region of target gene and activates transcription ; DNA | TRANSCRIPTION RNA 19 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Intracellular signaling pathways 20 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Complexity of signal transduction Integrins EXTRACELLULAR FLUID CYTOPLASM ■ Plasma membrane 0 Reception Q Transduction 0 Response Receptor , Shi-fc£ Signaling molecule Activation of cellular response vs Relay molecules in a signal transduction pathway Ion Channels [Ca*-] 1 / SRF c-Fos ERa Nur77 MITF C/EBPß Stat1/3 nm^c Pa*6 c"Fos HJstone HMGN1 ETV1 TIF1A ATF4 Mytl Mad1 YB1 Transcription Response 21 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Receptors Signal transduction Effectors Complexity of signal transduction Primary transduction of the signal Relay of signaling proteins - often function as molecular switches Amplification and processing of the signal Some signaling proteins shared -crosstalk of signaling pathways Outcome: One signaling molecule can induce many effectors extracellular signal molecule receptor protein „ plasma membrane SCAFFOLD II I III II •••••••• I V 1 < ft nuclear envelope activated effector protein NUCLEUS PRIMARY TRANSDUCTION RELAY TRANSDUCE AND AMPLIFY INTEGRATE SPREAD ANCHOR MODULATE signal response \ element GENE TRANSCRIPTION activatedgene ™™" 22 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Molecular switches -Signaling proteins often switch between "on" & "off" states -Signal turns signaling proteins active: phosphorylation/GTP-binding ■ This might lead to deactivation of other signaling proteins (counterbalance between pathways) SIGNAL IN protein protein kinase phosphatase SIGNAL OUT Protein kinase covalently binds phosphate group Protein phosphatase catalyze dephosphorylation SIGNAL IN GDP GTP GTP binding hydrolysis SIGNAL OUT Signal triggers exchange of GDP for GTP Hydrolysis of GTP inactivates the signaling protein (A) SIGNALING BY PHOSPHORYLATION (B) SIGNALING BYGTP-BINDING 23 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Major classes of cell-surface receptors 1) 2) 3) ION-CHANNEL-COUPLED RECEPTORS G-PROTEIN-COUPLED RECEPTORS ions signal molecule -i plasma -J membrane signal molecule inactive inactive inactive receptor G protein enzyme ENZYME-COUPLED RECEPTORS activated receptor and G protein signal molecule in form of a dimer OR inactive catalytic domain active catalytic domain activated' enzyme activated G protein ■ signal molecule Regulate ion-channel gating Control activity of enzymes or channel gating Catalytic activity Most commonly transmembrane proteins with protein kinase activity 24 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Ion-channel coupled receptors e.g., Chemical synapse -Conversion of chemical signal (neurotransmitter) to electric signal (action potential) at the postsynaptic neuron 25 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) i) Action potential arrives at axon terminal. (3) Ca2* entry causes neurotransmitter-contam i ng synaptic vesicles to release their contents by exocytosis B\ ___^Synaptic ^^yř vesicles (2) Voltage-gated Ca2* channels open and Ca2* enters the axon terminal. Neurotransmitter diffuses across the synaptic cleft and binds to ligand-gated ion channels on the postsynaptic membrane. Postsynaptic neuron V Localized membrane potential Neurotransmitter +++++- Ion movement Cytoplasm Open ligand-gated ion channel © 5) Binding of neurotransmitter opens ligand-gated ion channels, resulting in graded potentials. -4 ' Enzymatic ?,nu*on Reuptake by degradation^ from synapse presynaptic w f ) neuron ^-^ [&) Reuptake by the presynapic neuron, enzymatic degradation, and diffusion reduce neurotransmitter levels, terminating the signal. G-protein coupled receptors (GPCRs) - Largest family of cell-surface receptors, hundreds of members -Various signals: hormones, neurotransmitters, local mediators, light... - Heptaspan (7x) transmembrane proteins - Evolutionary conservative - sensory receptors across eukaryotic organisms - Smell (olfactory receptors) and vision (rhodopsin) in vertebrates - Intracellular domains binding G protein 26 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) fl Heterotrimeric structure of G proteins -GTP-binding protein (G protein) - three protein subunits - a (GDP/GTP binding), p, Y - Inactive state: all subunits bound together, GDP at a subunit i_i inactive G protein 27 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) G protein activation by GPCR - Ligand binds to GPCR -Conformation change and interaction with G protein - GTP replaces GDP and binds to a subunit -G protein separates into a subunit and ßY complex: both freely diffuse in plasma membrane and relay signal to target molecules 28 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) receptor protein inactive G pfoteio Short-term action of G proteins - a subunit exhibits GTPase activity: GTP hydrolyzed to GDP - a subunit and (3y complex re-form inactive trimeric G protein -a subunit GTPase activity regulates the signaling window Regulators of G protein signaling (RGS) promote GTPase activity = reduce the time available for activation of target molecules Active GPCR Inactive 29 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) G protein targets 1. Ion channels 2. Enzymes responsible for the production of second messengers: - Adenyl cyclase: cyclic AMP (cAMP) - Phospholipase C: hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG) 3. Rho GEFs: activate Rho GTPase Effectors AdenyLyl cyclase, ŤcAMP, PKA PI3K, adenylyl cyclase, 4-cAMP PLCp, tCaz+, PKC, Rho GTPases Rho GTPases 30 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) G protein-gated ion channels e.g., muscarinic acetylcholine receptors regulating potassium ion channels in heart muscle cells Muscarinic ACh receptor p-Adrenergic receptor Muscarinic acetylcholine receptor Slow depolarization = decreased heartbeat GIRK K+ channel Fast kinetics High concentration of G(3y VS. (3-adrenergic receptor •'Si 31 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Ca2+ channel (not G protein-gated) opens more readily = heart rate increases Slow kinetics Low concentration of G(3y Channel activation Closed G protein-regulated enzymes -Complex response: production of second messengers ■ Small molecules that easily diffuse in the cytosol, and initiate and coordinate intracellular signaling pathways NH2 Adenyl cyclase . , , r m inhibited by inhibitory G protein (Gi/ai) enylyl p-p - cAMP from ATP (rapid synthesis) Sfö P-O OH -cAMP removed by phosphodiesterase „ 0 y«* J 1 1 phosphodiesterase I j*2 with constitutive activity (rapid removal) x <£) -Q-p-o-CH 32 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) cAMP activates cAMP-dependent protein kinase (PKA) ■ GPCR -> Gs -> adenyl cyclase cAMP PKA activated adenylyl cyclase 33 F3i1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) cAMP: fast response vs slow response - Adrenalin PKA: metabolic enzymes (e.g., promotes glycogenosis), ion channels (e.g., increases heart rate) Glucagon (liver) or epinephrine (muscle and liver) Adenylate \ cyclase kinase A kinase A N Phosphorylase /-"^-^Phosphorylase Glycogen ,»-~>^ Glycogen kinase kinase synthase synthase 1 Phosphorylase s-^Phosphorylase (imctiv.) b a \ Glycogen,, > Glycogen,, _, Glucose 1 phosphate PKA phosphorylates cAMP responsive element-binding (CREB) protein ^ altered gene transcription element (CRE) GENE TRANSCRIPTION 34 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Epinephrine-stimulated cAMP synthesis resting state epinephrine / / beta-adrenergic receptor cAMP = second messenger extracellular surface cell membrane stimulated state .epinephrine beta-adrenergic receptor G-protein dissociation intracellular surface adenylyl cyclase extracellular surface 1 cell membrane j intracellular surface adenylyl cyclase cAM P x increased heart rate dilation of skeletal muscle blood vessels breakdown of glycogen to glucose cyclic AMP inactive PKA < 1 regulatory inactive subunit catalytic subunit v complex of cyclic AMP and regulatory subunits active catalytic subunits 35 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) G protein-regulated enzymes Phospholipase C - PIP2 into second messengers: DAG and IP3 - DAG recruits PKC - IP3 diffuses in the cytosol to open ER Ca2+ channels - Ca2+ serves as another second messenger: activation of PKC -downstream signaling 1 signal molecule 1 activated GPCR activated PI 4,5-bisphosphate [PI(4,5)P2] phospholipase C-p / diacylglycerol activated G protein lumen of endoplasmic reticulum activated protein %% kinase C ••Ca2+ open IP3-gated Ca2+-release channel 36 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Ca2+as a signaling molecule Ca -pump in ER membrane Ca -binding molecules in cytoplasm Concentration in the cytosol very low vs. extracellular space some cellular compartments (mainly ER, mitochondria) Concentration gradient maintained by specific membrane pumps & transporters Opening of the Ca2+ ion channels: fast influx (extracellular) or release (ER) into the cytosol and Na+-d riven Ca2+ exchanger [Ca^lJ-IO^M active Ca import in mitochondrion plasma membrane 37 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) CaM-kinases (Ca2+/calmodulin-dependent kinases) -Calmodulin serves as an intracellular Ca2+ receptor - Binding of Ca2+ (>2 ions; 4 sites in the structure) -> active conformation -> calmodulin binds to other proteins and affects their activity: serine/threonine CaM-kinases -Active CaM-kinases phosphorylate gene regulatory proteins 38 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Enzyme-coupled receptors Transmembrane proteins Upon ligand binding: Cytosolic domain -> activation of catalytic activity or association with enzymes RTKs represent the major group of these receptors RECEPTOR GUANYLYL CYCLASES RECEPTOR SERINE/ THREONINE KINASES RECEPTOR TYROSINE KINASES (RTKs) TYROSINE-KINASE-ASSOCIATED RECEPTORS N N Hi Mil iliU Guanylyl cyclase domains ANP receptor Ligand N N 1 Serine-threonine kinase domai insv^- Type II This is the kinase that phophorylates downstream TGF-|i effectors, receptor Ligand — m N^ ^N Extracellular space S -S-S- S S S 1 HI ■I KOTS Ä!M!ffl Mi c c Tyrosine kinase domains Cytosol NGF receptor Insulin receptor N N TfffflpifU JAK JAK C C Tyrosine kinase domains IL-6 receptor 39 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Receptor tyrosine kinases (RTKs) -Transmembrane glycoproteins - Extracellular ligand binding domain - Single transmembrane domain - Cytosolic tyrosine kinase domain - Ligands: growth factors = mitogenic signaling (cell cycle) - Ligand binding —► dimerization —► autocatalytic cross-phosphorylation 40 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) fibronectin-type Illlike domain plasma CYTOSOL membrane EG F insulin NGF FGF Eph receptor receptor, receptor I receptor I receptor IGF1 PDGF VEGF receptor receptor, receptor MCSF receptor signal molecule CYTOSOL inactive RTKs cross-phosphorylation by activated kinase domains Reminder: mitogenic signaling (overcoming the restriction point) Mitogen i Cyclin D Signal transduction pathway Helps to initiate cell cycle progression CDK4 / Cyclin D EG F Guanine nucleotide exchange factor p p Receptor tyrosine kinases MAP kinase kinase p p /|\S Cyclin D Transcription Phosphorylation Myc i Cyclin E í 41 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Signal transduction from activated RTKs - Phosphotyrosines serve as docking sites for: -Signaling proteins: activated by direct binding to the RTK (conformational change or phosphorylation by RTK) - Docking/adaptor proteins: attract other signaling proteins to the membrane where they are then activated - Binding through Src homology region 2 (SH2) or phosphotyrosine-binding (PTB) domains 42 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Signal transduction from activated RTKs growth factor molecules SIGNALING PROTEINS RELAY SIGNAL DOWNSTREAM Each RTK = plethora of docking sites: downstream signaling depends on the cell type and cellular context Signaling proteins directly e.g., phospholipase C gamma (cAMP); Src kinase, PI3K Via adaptor proteins: e.g., SOS (activating Ras) Sos 43 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Ras family of proteins RTK -> adapter proteins -> SOS (son of sevenless; = GEF) ^ active Ras plasma membrane INACTIVE Plasma membrane-bound monomeric GTPases (small G proteins) Activity stimulated by Ras guanine nucleotide exchange factors (GEFs) and inhibited by Ras GTPase-activating proteins (GAPs) promoting GTP hydrolysis ACTIVE Sos 44 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) MAPK cascade activated by Ras Canonical series of phosphorylation of protein kinases - Downstream mitogen activated protein kinase (MAPK) phosphorylates target proteins - MAPK kinase (MAPKK) phosphorylates MAPK - MAPKK kinase (MAPKKK) activated by Ras, phosphorylates MAPKK Signal amplification during signal transduction One ligand molecule activates a great number of effector proteins plasma membrane \_ MAP kinase kinase kinase (RafW - CYTOSOL 7TV active Ras protein i p |c fir MAP kinase kinase (Mek) - p p ADP MAP kinase (Erk) ADP p — protein X p — protein Y p — gene regulatory protein A p - gene regulatory protein B changes in protein activity J L J changes in gene expression 45 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Ras-Raf-MEK-ERK pathway ■ Key mitogenic signaling pathway: cell cycle progression through restriction point - principally by upregulating cyclin D expression - Promotes cell proliferation, growth and survival - Ras-ERK signaling important for differentiation during embryonic development Hyperactive Ras-ERK signaling - RASopathies - hereditary developmental syndromes, germline mutations in genes encoding RAS-ERK pathway proteins, e.g., Neurofibromatosis type 1 (NF1) - Associated with increased risk of cancer - Cancer: Ras proto-oncogenes mutant in more than 30% of cancers 46 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) Integration and crosstalk of signaling pathways -Cytosolic kinases and enzymes are regulated by several pathways -Crosstalkand integration of signals - context-dependent processing Protein activation achieved by additive effects of two (or more) pathways ■ Subunits activated by different pathways 47 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022) G protein G protein —*■ phospholipase C Grb2 PI 3-kinase uJUJUJ.Uocomic9ls.com 48 Bi1700en Cell Biology / 08 - Cell signaling (27 Apr 2022)