Bio-Signaling - Lehninger Chapter 12

1. Bio-Signaling - Lehninger Chapter 12 • 12.1 General Features of Signal Transduction • 12.2 G Protein–Coupled Receptors and Second Messengers • 12.3 Receptor Tyrosine Kinases • 12.4 Receptor GuanylylCyclases, cGMP, and Protein Kinase G • 12.5 Multivalent Scaffold Proteins and Membrane Rafts • 12.6 Gated Ion Channels • 12.7 Integrins: Bidirectional Cell Adhesion Receptors • 12.8 Regulation of Transcription by Steroid Hormones • 12.9 Signaling in Microorganisms and Plants • 12.10 Sensory Transduction in Vision, Olfaction, and Gustation • 12.11 Regulation of the Cell Cycle by Protein Kinases • 12.12 Oncogenes, Tumor Suppressor Genes, and Programmed Cell Death

2. 12.1 Molecular Mechanisms • Specificity - Receptors bind specific ligands and analogs, • toxins, inhibitors are Antagonists • Synthetic activators are Agonists • Affinity can be measured in crude preparation • Cooperativity can make the Receptor response curve steeper • Amplification by a cascade of enzyme activation events (often phosphorylation) • Desensitization - negative feed back • often limits the duration of the Signal response • Allows recalibration of the sensitivity of the response to continued stimulation • Integration of multiple signals through common intermediates, co-localization

4. Binding Affinity, Specific Binding is Saturable

5. 12.1a Scatchard analysis; Linear transformation of binding data • Old fashioned, statistically flawed • Receptor + Ligand ⇔ RL • Kd = [R][L]/[RL] • Separation of Bound (RL) and Free (L) Ligands • Plotting Bound ([RL]) vs [L]) total gives a hyperbolic plot with RLmax = Rtotal • (only if no nonspecific binding) • Plotting Bound/Free vs. Bound ([RL]/[L] vs [RL]) is a Scatchard Plot • Kd = (RT - [RL]) [L]/[RL] • [RL]/[L] = (RT - [RL])/Kd • Rearranging to the form y = mx + b • Gives: [RL]/[L] = -1/Kd ([RL]) + (RT/Kd)

7. 12.2 G-Protein Coupled Receptors • Receptors have 7 transmembrane helices • Interact with heterotrimeric G-Proteins • Gα binds GDP at rest • Gβ and Gγ complete the hetero-trimer • Activation of receptor leads to: • GDP release and GTP binding to Gα • Dissociation of Gβ and Gγ • Slow hydrolysis of GTP to GDP returns the system to the resting state

8. G-proteinsGTPase Cycle

9. 12.2a The β-Adrenergic Receptor (βAR) • Binds epinephrine (Adrenaline) Kd (5μM) • Agonist Isoproterenol Kd (0.4μM) • Antagonist Propanolol Kd (0.005μM) blocks activation • Gs.GTP activates adenylyl cyclase (AC) • AC make cAMP from ATP • cAMP binds to Protein Kinase A (PKA) regulatory subunits • PKA kinase subunits initiate kinase cascade • Desensitization • β&gamma binds to βAdrenergic Receptor Kinase (βARK) • βARK phosphorylates Serines on βAdrenergic Receptor • βAR-P binds βarrestin • preventing rebinding of GαS • Stimulating endocytosis • βarrestin also serves as a scaffold for MAPK cascade

10. The β-adrenergic pathway

11. β-adrenergic pathway, and desensitization

12. 12.2b Second Messengers • cAMP produced by AC; AC is regulated by many receptor pathways • Somatostatin Receptor activates Gαi inhibits AC • Distinct effects can be obtained by localization to scaffolds • Phosphatidylinositols => DAG + Inositol phosphates • Gαq.GTP activates Phospholipase C • PLC cleaves PIP2 to DAG + IP3 • IP3 activates Ca2+ channels on the ER • Increased [Ca2+] + DAG activate Protein Kinase C • PKC initiates yet another kinase signalling cascade • Ca2+ also binds to Calmodulin • Calmodulin binds to many targets including CamKinases

13. Second Messengers • Cyclic AMP (cAMP) activates PKA • Adenylate Cyclase: ATP -> cAMP + PPI • DAG and IP3 • Phospholipase C: PIP2 -> DAG + IP3 • Calcium • Release from ER, • Binds Calmodulin, CaM kinases

14. 12.3 Receptor Tyrosine Kinases • Receptor Tyrosine Kinases (RTKs) • activate intracellular kinase activity upon binding extracellular ligand

15. 12.3a Insulin Signal Transduction Cascades Insulin Promotes Cell growth, Glucose uptake and storage • Insulin Receptors (IR) bind 2 insulin peptide with 2 α chains • β chains (auto-)phosphorylate each other • P- β subunits now active tyrosine kinases • Active RTK initiates a signal transduction cascade • RTK phosphorylates Insulin Receptor Substrate-1 (IRS-1) • Adaptor proteins Grb2 and Sos bind to P-Tyr-IRS-1 via SH2 domain • Sos activates Ras GTPase • Ras.GTP activatesProtein Kinase Cascade • Ras activates Raf-1 kinase (MAPKKK) • Raf-1 kinase activates MEK kinase (MAPKK) • MEK kinase activates ERK kinase (MAPKinase) • ERK kinase activates Elk1 transcriptional activator

16. Activation of glycogen synthase by insulin • P-Tyr-IRS-1 binds Phosphoinositol 3 kinase (PI3K) • PI3K converts PIP2 + ATP => PIP3 • PIP3 activates protein kinase b (PKB) • PKB phosphorylates Glycogen Synthase Kinase (GSK) to INACTIVATE • GSK can no longer phosphorylate Glycogen Synthase • Unphospohrylated GS makes more glycogen • PKB also promotes vesicle fusion to place more GLUT4 transporters in the plasma membrane

18. Receptors coupled to JAK-STAT cascades • RTK ligand binding stimlates kinase • Janus Kinase (JAK) • JAK + ATP -> JAK-P + ADP • Signal Transducers and Activators of Transcription (STAT) • P-STAT form Dimers • Transported to nucleus • Bind to DNA • Activate transcription

19. Connections between signaling pathways

20. Receptor Guanyl Cyclases

21. 12.5 Scaffolding and Membrane Rafts • Up to 3500 signalling genes (> 500 kinases) in the human genome • Pre-assembled complexes can coordinate signalling pathways

22. Assembly through specific protein binding modules/domains • SH2 and PTB domains bind phospho-Tyrosine • SH3 domains bind Proline rich target peptides • Plextrin Homology (PH) domains bind PIP3 • Scaffolding and adaptor proteins are multivalent

23. Membrane Rafts and Caveolae can further enhance or insulate signalling pathways • Membrane associated signaling complexes may be pre-assembled in specialized bilayer domains • Signaling partners limited in isolated rafts

24. PTB domains can facilitatesubstrate assembly or auto-inhibition

25. 12.6 Gated Channels • Control Transmembrane Potential • Voltage Gated • Ligand Gated

26. Neuronal Action Potentials • At rest K+ channel is open; • K+in > K+out Δψ = -60 mV • Acetylcholine receptor channels open upon binding ligand • Na+ and Ca2+ can pass to cell interior membrane depolarizes • Prolonged ligand binding result in ligand-bound closed state • Acetyl choline is hydrolyzed by acetylcholinesterase in synapse • Voltage gated Na+ channels open (Na+ out => in) upon depolarization (amplify the signal) • Voltage gated K+ channels open (K+ in => out) membrane repolarizes locally • Wave of depolarization and repolarization (action potential) transmitted along the axon to the next synapse • Voltage gated Ca2+ channels allow Ca2+ influx • Ca2+ triggers SNAP/SNARE membrane fusion event that releases acetylcholine from exocytotic vesicles into the synapse

27. 12.11 Cell Cycle • Cells grow and divide according to an irreversible 4-stage program • G1 - S (DNA synthesis) - G2 - M (Mitosis and cell division) • DNA replication and Cell division are controlled by cyclin dependend kinases • Cyclins (regulatory subunits) are synthesized and degraded at specific points in the cell cycle • Progress through the cell cycle is highly regulated by • Extracellular signalling pathways (growth factors) • Internal surveillance, cycle checkpoints for DNA damage, synthesis, etc

28. Four irreversible stages • G1 • S (DNA synthesis) • G2 • M (Mitosis and cell division)

29. CDKs Activated in complex with Cyclins

30. Cyclin Synthesis and Degradation controls cell cycle transitions

32. Internal regulation of Cell Cycle Checkpoints • Tumor Suppressor Genes P53 and retino blastoma halt cell cycle progression in the case of DNA damage

33. 12.12 Cancer and Apoptosis • Cancer is inevitable because of 2 laws • Survival of the fittest • Murphy's law -- Anything that can go wrong - will. • Cell growth in a multi-cellular organism is mostly halted in adults • Many redundant signaling pathways can activate or suppress growth in different cells • Mutations in signaling pathways can lead to uncontrolled growth • Cells that grow out-compete, and interfere with normal cells

34. Cancer Genes • Oncogenes • gain of function mutations • Activate cell growth constitutively (dominant) • Proto-oncogenes normally activate growth, cell cycle only when stimulated • Tumor Supressor genes normally controll cell growth • Mutations lead to loss of function (recessive) • Both genes need to be inactivated to observe the cancer phenotype

36. 12.12b Apoptosis • In addition to growth suppression cells have an active suicide program • Triggered by immune system responses • Cell cycle checkpoint repeated failures • Protease cascade of caspases