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signal transduction.docx

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Richard B Day

February 26 , 2013 Biology 2B03: Cell Biology Signal Transduction Signal Transduction - what is signalling  change in behaviour/change in response of the cell  requires receptors: signal must be interpreted in some way  signal might be received by different cells and elicit different response  cell-cell signalling: transmitting information from one cell to another and inducing a change in behaviour )response) Cells Communicate By - direct cell-to-cell contact, junctions: quickly transmit information - cell-to-cell via plasmodesmata plants - extracellular signalling molecules: cells that are far apart communicate through secreting signal Signaling Molecules - produced by signalling cells - induce a specific response in target cells with receptors for ligand - can be proteins Responses include - changes in gene transcription, cell division, growth, differentiation, changes in shape, movement, changes in metabolism Signal Transduction - process of converting extracellular signals into a cellular response; interpreting the signal (a) fast response: changes in enzyme activation, all you do is modify a protein that already in the cell (b) slow response: changes in gene transcription, many things have to happen before you can see an overall change at the cellular level Signaling Molecules and Cell-Surface Receptors - signalling cells: source of signal or ligand - target cells: receptors that receive signal - ligand & receptor: specificity and affinity are determined by molecular complementarity - signal inducing cellular response = signal transduction pathway Types of Intercellular Signaling - endocrine signalling:  all cells have the potential to respond however only those which have the target will respond  signalling molecule: hormone (soluble extracellular molecules)  target: distant - paracrine signalling:  signalling molecule: e.g. growth factors, neurotransmitters (soluble extracellular molecules)  target: proximate Other Proximal Signaling - integral membrane proteins:  signalling molecule: membrane bound  target: neighbour - cell to cell signalling via plasmodesmata in plants  due to the lack of cytokinesis in mitosis of plant cells  target: neighbour  estabilishment of vascular system  gap junction direct cell-cell contact in animal cells Autocrine Signaling - signalling molecule: growth factors (disruption may lead to tumorigenesis) - target: self - soluble extracellular molecules - cells respond to signals they produce themselves - e.g. growth factor Mechanisms of Signal Transduction are Highly Conserved - signal synthesis - release - transport - receptor binding – activation - initiation of a signal transduction pathway (STP) - changes in cellular function 0 two major types of responses:  changes in protein activity  change in protein levels (via transcription)(there amy be more than one response) - removal of signal The Specificity of a Signal Response is Achieved by - ligand binding specificity: binding to receptor  a receptor generally binds only one signalling molecule or a small family of closely related molecules - effector specificity: intracellular response  there is a unique cellular response to ligand-receptor association on a specific cell; some signalling molecules bind to receptors Ligand Binding & Effector Specificity - signalling molecule = ligand or 1 messenger - ligand binding to receptor: molecular complementarity  induces conformational change in receptor  initiates a cascade of reactions  produces cellular response - single changes can have very dramatic effects Cellular Response may not Require Binding to all Receptors - how does this happen?  Typical cell has 10 to 10 receptors  K d dissociation constant of receptor-ligand complex  Also [L] where half receptors occupied  Lower the K , the higher the affinity d  Ligand [L] needed to induce maximal cell response is lower than amount needed to saturate receptors  Implies that there’s an amplification response that occurs Seven Major Classes of Cell-Surface Receptors - differ in morphology of the receptors - largest class = GPCR February 28 , 2013 Types of Cell Surface Receptors - different ways to realy a signal - G-protein coupled receptors:  Ligand binding activates G protein inside the cell  Causes activation of 2 enzyme (effector)  Second messengers generated - Receptor tyrosine kinases:  Ligand causes receptor dimerization, single transmembrane receptors  Phosphorylates self & other substrates and changes their activity - tyrosine kinase-linked receptors = cytokine receptor  ligand causes receptor dimerization  dimer now interacts & activates cytosolic kinase  phosphorylation can change activity of a cellular protein - types of enzymes in signalling pathways: protein kinases and GTPase switch proteins Signaling Pathways Objectives - describe key steps in signal transduction pathways:  cytokine receptor and JAK-STAT  receptor tyrosine kinase (RTK) and Ras  G-protein coupled receptor (GPCR) - identify similarities and differences between these pathways - predict and interpret the effects of mutations in signal transduction pathways (STP) Example 1: Signalling Growth and Differentiation: Formation of Red Blood Cells - cytokine receptors and JAK-STAT pathway  cytokines (signal): small secreted signal proteins  want to increase the production of red blood cells  erythropoieting (Epo) signals erythroid progenitor cells in the blood marrow to reproduce. Without Epo erythroid progenitors undergo apoptosis (cell death)  id there is enough O 2he pathway stops  leads to an increase in RBCs  low O 2evels cause synthesis and release of Epo resulting in the production of red blood cell levels  how is low O 2etected? Oxygen sensitive transcription factor in kidneys Cytokine Receptor and JAK-STAT - ligand: cytokine, Epo - receptor: Epo receptor (EpoR) - intracellular transduction: JAK kinases and STAT transcription factors - cellular response: transcription of STAT target genes - because one ligand attaches to two receptors they are indirectly connected (dimerization) switching protein into an active state - dimerization and phosphorylation are key steps in activation if the cytokine pathway  phosphorylation: conformational change in activation lip of JAK kinase that increases enzyme activity. Phosphorylation of tyrosines on EpoR occues  JAK kinase have a weak kinase activity: cannot go on to phosphorylate but can phosphorylate itself, so when to JAK kinases are next to each other it is enough to phosphorylate each other  Phosphate comes from ATP and produces ADP as by product  JAK kinases are now strong and can phosphorylate the intracellular domain of the receptor - Phosphorylation of tyrosines on EpoR at docking site allows binding of the STAT transcription factor to Phos-EpoR:  Docking sites can bind any protein that contains Sh2 domain, when they are phosphorylated  STAT transcription factor is now docked and the kinase can phosphorylate the STAT protein  Leaves the docking site and can bind together due to protein-protein interaction domains  We have created a transcription - STAT is phosphorylated allowing dimerization and nuclear localization sequence (NLS) is exposed SH2 Domain: Recognizes Phosphorylated Tyrosine - Protein-protein interactions - P-Tyr containing peptide: Pro-Asn-pTyr-Glu-Glu-Ile-Pro - Backbone is yellow - R-groups are green - Phosphate is blue - Tight fit (molecular complementarity) is only achieved when tyrosine is phosphorylated: P-Tyr and Ile fit into binding pockets Protein-Protein Interactions: Also Based Upon Molecular Complementarity - Protein interaction domains - Domains identified that recognize specific sequences on their target proteins. - Protein interactions are required for the assembly of signalling complexes  E.g. (A)SH2 domains recognized phosphor-tyrosine (C) PDZ domains recognize hydrophobic C-terminus Cytokine Receptors and JAK-STAT - Target genes of STAT transcription factors? - In eruthroid progenitors, STAT5 increases transcription of Bcl-x whicL prevents death of these cells; instead they become mature erythroid cells (production of erythrocytes) - We can see an accumulation of red blood cells in the kidney - Embryo is smaller and we do not observe a red stain - Not seeing red cell production in the absence of the receptor - Embryo did develop (same morphological characteristics) by simply using the nutrients provided by the mother - Epo receptor is necessary for the production of red blood cells Turning Off the Signal: Examples from EpoR Pathway - Can experience blood clotting as there are too many blood cells as they start to aggregate in your vessels causing heart attack or stroke - Training at higher elevation: lower partial pressure, the body responds by making more red blood cells - Blood doping - Inject Epo - SHP1 Phosphatase – short term regulation:  Reversing kinase activity (turning off kinase)  SHP1 has SH2 domain and can bind to phosphorylated tyrosines and dephosphorylates the JAK kinase  Turns of the receptor and shuts off the pathway  Can be reversed fairly quickly - Protein degradation by SOCs – long term regulation  Don’t want to produce any more blood cells for an extended period of time  SOCs protein has an SH2 domain and can bind to phosphorylated binding sites  Turns off pathway quickly as it blocks all the dock binding sites  Part of these SOCs proteins are E3 ubiquitin ligase  Targets the JAK protein for degradation  Turn off pathway by removing JAK kinase  Once JAK kinase is removes it facilitates the dimerization of the two proteins  In order to turn the pathway back on we need to make more of the JAK protein through transcription - We can get rid of the ligand, through exocytosis it can be put back on to the receptor Example 2: G Protein-Coupled Receptors (GPCR) - seven-membrane spanning:  7 transmembrane α-helices  4 extracellular segments (E1-4)  4 cytoplasmic segments (C1-4) - linked to small, trimeric G-proteins Family Includes - signalling responses to stress - light-activated rhodopsins in eye - odorant receptors in mammalian nose - hormones & neurotransmitter receptors - plant growth hormone receptors - the recent discovery of a glucose-sensing GPCR system in Saccharomyces cerevisiae A Typical GPCR Mediated Pathway: Stress Response - e.g. adrenergic receptors - signal: catecholamines: epinephrine, norepinephrine - receptor: GPCR - intracellular transduction: adenylyl cyclase - effector enzyme/second messenger: cAMP - cellular response: release of stored energy  fast: enzyme activation  slow: activation of transcription Adrenergic Receptors - two types of receptors α2 and β - epinephrine has different effects on different tissues used in the fight or flight response - β-adrenergic receptors are stimulatory:  Liver & adipose cells: glycolysis and lipolysis  Heart muscle: increase contraction, increase blood supply to tissues  Smooth muscle cells of intestine: increase relaxation, save energy for major locomotary muscles - α2-adrenergic receptors are inhibitory:  Blood vessels of smooth muscle of intestine, skin, kidney: cause arteries to constrict, blood supply is reduced to periphery - Some may have a combination of both - All of these cells have the potential to respond in different ways to the same signal (epinephrine) accomplishing a coordinated task - Net result: increased energy for rapid motion in response to stress or exercise March 4 , 2013 Catecholamines - products of adrenal gland - epinephrine = adrenaline - binds 2 types of GPCR (α2 and β) - mediates stress response to fright or heavy exercise  increased need for ATP  breakdown of glycogen and triacylglycerols to  glucose in liver: glycosis  fatty acids in adipose tissue: lipolysis - norepinephrine: secreted by nerve cells, acts as neurotransmitter for flight response GPCR - Membrane-associated via lipid - Gα= GTPase switch protein:  Active, when bound to GTP  Inactive, when bound to GDP Activation of Receptor - Binding in the extracellular domain will result in a change in the intracellular conformation allowing it to interact with the G-protein Activation of Effector - only active as long as the G-protein is bound to it - guanine exchange:  release of GDP  GTP binds  Cystolic [GTP] high - GTPase; fast - Depends on hydrolysis of GTP for rate β-adrenergic Receptors - coupled to stimulatory G proteins, Gs - three subunits α, β, γ - alternate between active, G -GTPsαound and inactive G -GDP bound sα - G sαTP, dissociates from G , theβγbinds and activates adenylyl cyclase = Increase cAMP - Intrinsic G sαTPase hydrolyzes GTp to GDP, so G -GDP reasssαiates with G βγthus inactivating Adenylyl Cyclase = cystolic decrease in cAMP α2-adrenergic Receptors - same G anβ G subuγits as β-adrenergic receptors - different G ,αinhibitory G iα - inhibits adenylyl cyclase: no increase in cAMP (net decrease) - interacts with different region of Adenylyl cyclase catalytic domain - activated in response to adenosine and prostaglandin (PGE ) 1 Different Ligand-Receptor Complexes Stimulate and Inhibit Adenylyl Cyclase - allows fine tuning of cAMP levels in a particular cell What is Adenylyl Cyclase? - increase cystolic [cAMP] - enzyme that takes ATP and converts it into cAMP - ultimate goal is to increase ATP for energy in the cell - we use little ATP to make a lot of ATP through the amplification of the signalling pathways Degradation of cAMP by Phosphodiesterases - cAMP is unstable in the cell - already in the cytosol and are always active convert cAMP into 5’-AMP - example: when the enzymes are turned off Ca is turned off - cAMP is naturally unstable: as soon as it’s made it can be degraded Second Messengers: cAMP - what are second messengers: binding of ligand leads to rapid but short-lived increase in cystolic concentration of low molecular weight, intracellular signalling molecules called second messengers - for example: cAMP - what do cAMP do?  Increased cAMP has different effects in different cell types  Many effects mediated by regulation the activity of an enzyme  cAMP-dependent protein kinases = Protein Kinase A’s (PKAs)  specificity determined by each PKA and its substrates in particular cell type  serine/threonine kinases  PKAs represent about 2% of human genome PKA - cooperative allostery = binding of first cAMP reduces the K fordbinding of the second - thus, small changes in [cAMP] can cause large changes in the [active PKA] What does PKA do? - what are the targets of PKA? - Epinephrine signal pathway: increased supply of energy to body in times of exercise or stress  Occurs in muscle & liver  Glycogen: major storage form of glucose; polymer of glucose  Synthesized by one set of enzymes (glycogen synthase) – turn off  Degraded by another set of enzymes (glycogen phosphorylase) – turn on  Epinephrine-stimulated increase in cAMP and activation of various PKAs in particular cells induces:  In muscle, glycogen to glucose-6-phosphate (G-6-P) o G-6-P enters glycolytic pathway metabolized to ATP, and provides energy for muscle contraction  In liver, enhances glycogen degradation to G-6-P o By inhibiting glycogen synthesis (phosphorylation and inactivation of glycogen synthase) o And stimulation glycogen degradation (indirect activation of glycogen phosphorylase that degrades glycogen) o G-6-P converted to free glucose, released to blood and transported to other tissues  Net effect: ATP for muscles and brain Medical Relevance - some bacterial toxins can chemically modify G -GTsαso that it is non- hydrolyzable - Gsαtays in activated state in the absence of adrenaline stimulation - Adenylyl cyclase stays active, cAMP level rise - For example:  Cholera toxin from Vibrio cholera bacteria  Enterotoxin from certain E. coli strains - increases cAMP: relaxation of gut, loss of water and electrolytes, watery diarrhea - benefit to pathogen, disperses it to other hosts Activation of Gene Transcription by G Coupled-Receptors - PKA-C to the nucleus. - Phosphorylation and activation of the transcription factor, CREB - Binds to CRE (cAMP response element) regulatory element of various target genes - Together bind to a promoter on a collection of other genes - Including genes for enzymes required for glucose production How can Signaling Cascades Amplify the Signal within the Cell March 5 , 2013 Example 3: Signalling Cell Proliferation, Cell Differentiation, Cell Survival/Apoptosis, Cell Metabolism - receptor tyrosine Kinases (RTKs) pathways and activation of Ras G-protein - involved in growth and differentiation in cell - induce cell to proliferate and divide - ligands/signals include hormones: nerve growth factor (NGF), platelet derived growth factor (PDGF) epidermal growth factor (EGF), insulin - activates intrinsic kinase activity of transmembrane cell surface receptors: receptor itself has kinase activity Form Signal Perception to Cellular Response - ligand: growth hormone - receptor: RTK - intracellular transduction:  adaptors: GRB2 – act as linkers  Ras proteins (GTPase)  Ras effectors = GEF and GAP – proteins that interact with Ras  Cascade of kinases  MAP Kinase – end product protein  Transcription factors - cellular response: transcription RTK Activation - RTK: monomeric, single pass, transmembrane receptor - Ligand binding induces dimerization of receptor EGF is hormone one molecule of EGF binds to receptor bringing about a change in conformation - The receptors can now dimerize together - Receptor autophosphorylation of tyrosine residues - Phosphotyrosines serve as docking sites for adapter proteins - Adaptors plus Ras protein couples RTKs to other components of signalling pathway - Weak kinases, when they diffuse and our brought close together they can auto phosphorylate and switch to an active state - Docking site once it is in the actiated conformation Role of Adaptor Proteins - proteins that contain protein-protein interaction domains (e.g. SH2, PTB), but do NOT have any enzymatic or signalling activity - couple RTKs to other proteins in pathway - scaffold proteins = many pro
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