Chapter 15: Signal transduction Know the terminology: Enzyme-linked receptor, G-protein linked receptor, nuclear hormone receptor, G-protein, adaptor protein,

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<ul><li> Slide 1 </li> <li> Chapter 15: Signal transduction Know the terminology: Enzyme-linked receptor, G-protein linked receptor, nuclear hormone receptor, G-protein, adaptor protein, scaffolding protein, SH2 domain, MAPK, Ras, protein kinase, MAPK, protein phosphatase, phospholipase, phosphodiesterase, cAMP, crosstalk, </li> <li> Slide 2 </li> <li> Chapter 15: Signal transduction Outline: General principles of signal transduction Overview of: Signaling Receptors Transducers Targets Major types of cell-surface receptors RTK signaling G-protein signaling </li> <li> Slide 3 </li> <li> General Principles of Signal Transduction 1. Communication usually involves a (i) signaling molecule, (ii) a receptor, (iii) intracellular signal transducers and (iv) targets </li> <li> Slide 4 </li> <li> General Principles of Signal Transduction 2. Each cell responds to a complex profile of signaling molecules (crosstalk) </li> <li> Slide 5 </li> <li> General Principles of Signal Transduction 3. Different cells respond differently to a particular signaling molecule </li> <li> Slide 6 </li> <li> General Principles of Signal Transduction 4. Cells can remember the effects of some signals 5. Cells can adjust their sensitivity to a signal </li> <li> Slide 7 </li> <li> General Principles of Signal Transduction 4. Cells can remember the effects of some signals 5. Cells can adjust their sensitivity to a signal </li> <li> Slide 8 </li> <li> General Principles of Signal Transduction 6. Signal can exhibit complex responses to signal concentration </li> <li> Slide 9 </li> <li> Signaling molecules Signaling molecules come in many chemical forms: Proteins: insulin, glucagon Steroids et al.: testosterone, estradiol, cortisol Amines: thyroxine, catecholamines, acetylcholine Gases: nitric oxide Signaling pathways require molecules with rapid rates of synthesis and degradation Typically released from one cell and recognized by another cell </li> <li> Slide 10 </li> <li> Signaling molecules Secretory signals: Autocrine-signals affect same cell or cell type Paracrine-signals affect neighbouring cell Endocrine-signals affect distant cells Contact-dependent signals: -signals are not released but affect other cells in contact through protein-protein interactions </li> <li> Slide 11 </li> <li> Autocrine signaling Signals released by one cell affect other cells in the immediate vicinity Amplify a response by inducing many like-cells to respond in the same way Allows cells to exhibit a coordinated response (a community effect) </li> <li> Slide 12 </li> <li> Autocrine signaling </li> <li> Slide 13 </li> <li> Paracrine signaling Signals released by one cell affect different cells in the immediate vicinity Synaptic transmission resembles paracrine stimulation but the response is limited to cells in very close proximity The outward propagation of the signal is limited by cellular uptake, extracellular degradation, and binding </li> <li> Slide 14 </li> <li> Endocrine signaling Signals released by one cell affect different cells far away Endocrine signaling often exerts multiple effects on the organism by affecting many different tissues </li> <li> Slide 15 </li> <li> Slide 16 </li> <li> Receptors Proteins that bind signals and initiate a signaling cascade Cell membrane receptors -integral membrane proteins that bind an extracellular signal and start a signal cascade Intracellular receptors -nuclear hormone receptors </li> <li> Slide 17 </li> <li> Nuclear hormone receptors Examples that we have already discussed include steroid hormone receptor and thyroid hormone receptor NHRs are transcription factors that respond to specific ligands Ligands alter the ability to bind to specific DNA regulatory elements </li> <li> Slide 18 </li> <li> Intracellular signal transduction Once the receptor is activated, the signal is propagated by proteins that act as: Relay proteins Messenger proteins Adaptor proteins Amplifier proteins Transducer proteins Bifurcation proteins Integrator proteins Latent gene regulatory proteins </li> <li> Slide 19 </li> <li> Slide 20 </li> <li> Intracellular signal transduction Activated cell membrane receptors can alter the activity of intracellular enzymes including: Protein modifying enzymes kinases/ phosphatases acetylases/ deacetylases Lipid modifying enzymes Phospholipases Phosphotidyl inositol kinase Nucleotide modifying enzymes cyclases/ phosphodiesterases </li> <li> Slide 21 </li> <li> Protein kinases </li> <li> Slide 22 </li> <li> Phospholipases PLC generates DAG and phosphoinositides, such as IP3 (inositol 1, 4, 5- triphosphate) </li> <li> Slide 23 </li> <li> Targets The final targets of signaling cascades are usually proteins: Regulators of gene expression (transcription factors, histone remodeling enzymes) Enzymes (metabolic enzymes) Structural proteins (cytoskeletal proteins) Effects alter activity (catalytic, DNA binding) or the ability to interact with other proteins (structural proteins, subcellular localization). </li> <li> Slide 24 </li> <li> Cell surface receptors 3 main classes of cell surface receptors: Ion-channel linked receptors Enzyme linked receptors may possess intrinsic enzyme activity or, once ligands bind, activate enzyme activity G-protein linked receptors are trimeric GTP- binding protein (G-protein) that regulate the activity of other proteins </li> <li> Slide 25 </li> <li> Enzyme-linked receptors 5 main classes distinguished by: type of effector (e.g. kinase vs. phosphatase) target (serine/threonine, tyrosine, histidine) type of linkage between receptor and enzyme 1. Receptor tyrosine kinase (-RTK) 2. Tyrosine kinase linked receptor 3. Receptor serine/threonine kinase 4. Receptor guanylyl cyclase 5. Histidine-kinase associated receptors </li> <li> Slide 26 </li> <li> Receptor tyrosine kinases Most common type of receptor for many common protein hormones including EGF, PDGF, FGF, HGF, IGF-1, VEGF, NGF. </li> <li> Slide 27 </li> <li> Receptor tyrosine kinases Receptor itself possesses intrinsic tyrosine kinase activity Once the ligand binds, the receptor can dimerize and it become an active tyrosine kinase It phosphorylates itself (autophosphorylation), causing: 1. Increase kinase activity 2. Increased affinity for other proteins Once bound, these docking proteins can become phosphorylated </li> <li> Slide 28 </li> <li> Ligand-dependent autophosphorylation and docking </li> <li> Slide 29 </li> <li> Slide 30 </li> <li> Docking of intracellular proteins on phosphotyrosines Phosphotyrosine domains are binding sites for many different proteins with SH2 (=PTB) domains These can be enzymes (e.g., PLC, PI3K) or they can act as adaptor molecules to bind other proteins </li> <li> Slide 31 </li> <li> Linking RTK to Ras and the MAPK cascade Once an adaptor protein (e.g., Grb2) binds to the RTK, it attracts another protein - Ras GEF (guanine nucleotide exchange factor) Ras GEF induces Ras to exchange its GDP for GTP (activating Ras). Active Ras then activates MAPKKK, which phosphorylates and activates MAPKK, which phosphorylates and activates MAPK, which phosphorylates many proteins, including transcription factors. </li> <li> Slide 32 </li> <li> Activation of Ras </li> <li> Slide 33 </li> <li> Activation of MAPK cascade </li> <li> Slide 34 </li> <li> Scaffolding proteins help organize MAPKs </li> <li> Slide 35 </li> <li> Summary: Enzyme-linked receptors How do enzyme-linked receptors generate variable cellular responses? Multiplicity of players (receptors, kinases etc) arise from gene duplication and divergence Recognize the critical role of phosphorylation/ dephosphorylation control as molecular switches Adaptor molecules allow construction of protein signaling cascades with variable outputs </li> <li> Slide 36 </li> <li> G-protein linked receptors Ligand: Diverse ligands, such as epinephrine Receptor: Integral membrane protein with 7- transmembrane domains G-protein: Trimeric protein attached to the cell membrane by lipid anchors Effectors: Target proteins that show altered activity when they interact with activated G- protein subunits ( or ) </li> <li> Slide 37 </li> <li> G-protein linked receptors and G-proteins Receptor G-protein </li> <li> Slide 38 </li> <li> Interaction between receptor and G-protein Once the ligand binds, the activated receptor recruits a G-protein Nucleotide exchange occurs (GTP replaces GDP) and the trimer dissociates into 2 parts: - subunit - subunit Both parts can regulate downstream pathways </li> <li> Slide 39 </li> <li> G-protein dissociation </li> <li> Slide 40 </li> <li> GTP hydrolysis ends signaling and induces trimerization </li> <li> Slide 41 </li> <li> G s proteins are stimulatory Upon dissociation, a G s protein stimulates an effector enzyme, such as adenylate cyclase Adenylate cyclase converts ATP to cAMP Elevated cAMP stimulates cAMP-dependent protein kinase (PKA) by inducing the release of inhibitory subunits </li> <li> Slide 42 </li> <li> PKA activation by cAMP </li> <li> Slide 43 </li> <li> PKA activates gene expression </li> <li> Slide 44 </li> <li> Inactivation of PKA pathway The G-protein -PKA pathway is inactivated by: Receptor desensitization GTP hydrolysis in G-protein cAMP hydrolysis by phosphodiesterase PKA inhibition Phosphatase action on PKA targets Activation of an antagonistic pathway (G i ) </li> <li> Slide 45 </li> <li> G-proteins and phospholipases Some G-proteins activate PLC (phospholipase C), triggering formation of inositol triphosphate (IP3) and diacylglycerol (DAG) </li> <li> Slide 46 </li> <li> DAG, IP3, Ca 2+ and signal transduction DAG: substrate for production of eicosanoids, potent signaling molecules including arachadonic acid activates PKC IP3: induces release of Ca 2+ from ER stores via IP3- sensitive Ca-channels Ca 2+ : Elevated Ca 2+ can activates PKC and CamK. </li> <li> Slide 47 </li> <li> Interactions between G-proteins and RTKs </li> </ul>