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Signal Transduction 2

Signal Transduction 2. BL4010 12.02.05. Signalling vocabulary. Signal/stimulus Effector Receptor Messenger Ligand Cascade. Heterotrimeric G Proteins. A model for their activity

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Signal Transduction 2

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  1. Signal Transduction 2 BL4010 12.02.05

  2. Signalling vocabulary • Signal/stimulus • Effector • Receptor • Messenger • Ligand • Cascade

  3. Heterotrimeric G Proteins A model for their activity • Binding of hormone, etc., to receptor protein in the membrane triggers dissociation of GDP and binding of GTP to -subunit of G protein • G-GTP complex dissociates from G and migrates to effector sites, activating or inhibiting • But it is now clear that G also functions as a signalling device

  4. GTPase activating proteins (GAPs)

  5. MAP Kinase Cascade

  6. MAP Kinase Cascade

  7. When signal molecule misbehave

  8. Phospholipases Release Second Messengers • Inositol phospholipids yield IP3 and DAG • PLC is activated by 7-TMS receptors and G proteins • PLC is activated by receptor tyrosine kinases (via phosphorylation) • Note PI metabolic pathways and the role of lithium

  9. Phospholipase targets

  10. Phosphotidyl inositols as secondary messengers

  11. Many different activators of phospholipase

  12. Phospholipase C isozymes • src-homology domains (SH) • SH2 mediates interactions with phosphotyrosinated proteins • SH3 interacts with cytoskeletal proteins

  13. Other Lipids as Messengers Recent findings - lots more to come • More recently than for PI, other phospholipids have been found to produce second messengers! • Phosphatidyl choline can produce prostaglandins, diacylglycerol and/or phosphatidic acid • Sphingomyelin and glycosphingolipids also produce signals such as ceramide, a trigger of apoptosis - programmed cell death

  14. Ca2+as a Second Messenger Several sources of Ca2+in cells! • [Ca2+] in cells is normally very low: < 1M • Calcium can enter cell from outside or from ER and calciosomes (plants store Ca2+ in oxalate crystals) • CICR - Calcium-Induced Calcium Release • see animation

  15. Calcium Oscillations! M. Berridge's model of Ca2+signals • Ca2+ was once thought to merely rise in cells to signal and drop when the signal was over • Berridge's work demonstrates that Ca2+ levels oscillate in cells! • The purpose may be to protect cell components that are sensitive to high calcium, or perhaps to create waves of Ca2+ in the cell

  16. Patch clamp

  17. Ca2+-Binding Proteins Mediators of Ca2+effects in cells • Many cellular proteins modulate Ca2+ effects • 3 main types: protein kinase Cs, Ca2+-modulated proteins and annexins • Kretsinger characterized the structure of parvalbumin, prototype of Ca2+-modulated proteins • "EF hand" proteins bind BAA helices

  18. Calmodulin

  19. Protein Modules in Signal Transduction • Signal transduction in cell occurs via protein-protein and protein-lipid interactions based on protein modules • Most signaling proteins consist of two or more modules • This permits assembly of functional signaling complexes

  20. Transduction of two second messenger signals PKC is activated by DAG and Ca2+ • Most PKC isozymes have several domains, including ATP-binding domain, substrate-binding domain, Ca-binding domain and a phorbol ester-binding domain • Phorbol esters are apparent analogues of DAG • Cellular phosphatases dephosphorylate target proteins • Read about okadaic acid

  21. Localization of Signaling Proteins • Adaptor proteins provide docking sites for signaling modules at the membrane • Typical case: IRS-1 (Insulin Receptor Substrate-1) • N-terminal PH domain • PTB domain • 18 potential tyrosine phosphorylation sites • PH and PTB direct IRS-1 to receptor tyrosine kinase - signaling events follow!

  22. Lipids Rafts • first hypothesized in 1988 • nice review: Cary, L. A. & Cooper, J. A. (2000) Molecular switches in lipid rafts.  Nature. 404, 945-947 • Moffett, S., Brown, D. A. & Linder, M. E. (2000) Lipid-dependent targeting of G proteins into rafts. J. Biol.Chem. 275, 2191-2198.

  23. Many actin binding proteins are known to bind to polyphosphoinositides and to be regulated by them • Activation of receptor causes reorganization of the rafts

  24. Simons, K. et al. J. Clin. Invest. 2002;110:597-603 J. Fantini, N. Garmy, R. Mahfoud and N. YahiLipid rafts: structure, function and role in HIV, Alzheimer’s and prion diseases Expert Reviews in Molecular Medicine: 20 December 2002

  25. Cells of Nervous Systems Neurons and Neuroglia (Glial Cells) • Neurons contain processes, including an axon and dendrites • Axon is covered with myelin sheath and cellular sheath, except at nodes of Ranvier • The axon ends in synaptic termini, aka synaptic knobs or synaptic bulbs • Three kinds of neurons: sensory neurons, motor neurons and interneurons

  26. Ion Gradients The source of electrical potentials in neurons • Nerve impulses consist of electrical signals that are transient changes in the electrical potential differences (voltages) across neuron membrane • Difference between Nernst potential and actual potential represents a thermodynamic push

  27. The Action Potential series of changes in potential that constitute a nerve impulse • Small depolarization (from -60 to -40 mV) opens voltage-gated ion channels - Na flows in • Potential rises to +30 mV, Na channels close, K channels open. K streams out, lowering potential

  28. Action potential • Action potentials flow along the axon to the synapse • Number and frequency important, not intensity

  29. Voltage-Gated Na, K Channels Clustered in Nodes of Ranvier • These channels are voltage-sensitive - voltage changes cause conformational changes and gating

  30. Some 7000 sodium ions pass through each channel during the brief period (about 1 millisecond) that it remains open.

  31. Streptomyces voltage gated K+ channel

  32. Communication at the Synapse A crucial feature of neurotransmission • Ratio of synapses to neurons in human forebrain is 40,000 to 1! • Chemical synapses are different from electrical • Neurotransmitters facilitate cell-cell communication at the synapse • Note families of neurotransmitters in Table 34.6

  33. The Cholinergic Synapse A model for many others • Synaptic vesicles in synaptic knobs contain acetylcholine (10,000 molecules per vesicle) • Arriving action potential depolarizes membrane, opening Ca channels and causing vesicles to fuse with plasma membrane • Acetylcholine spills into cleft, migrates to adjacent cells and binds to receptors • Toxin effects: botulism toxin inhibits Ac-choline release, black widow's latrotoxin protein overstimulates

  34. Two Classes of Ac-Ch Receptor Nicotinic and muscarinic • As always, toxic agents have helped to identify and purify hard-to-find biomolecules • Nicotinic Ac-Ch receptors are voltage-gated ion channels • Muscarinic Ac-Ch receptors are transmembrane proteins that interact with G proteins • Acetylcholinesterase degrades Ac-Ch in cleft • Transport proteins and V-type H+-ATPases return Ac-Ch to vesicles - called reuptake

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