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Signalling vocabulary

Signalling vocabulary

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Signalling vocabulary

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

  2. Types of Receptors • 7-TMS receptors (G protein receptors) • extracellular site for hormone (ligand) • intracellular site for GTP-binding protein • Single-transmembrane segment receptors • extracellular site for hormone (ligand) • intracellular catalytic domain - e.g. kinase or guanylyl cyclase • Oligomeric ion channels

  3. G-protein coupled receptors Receptors that interact with G proteins • Seven putative alpha-helical transmembrane segments • Extracellular domain interacts with hormone • Intracellular domain interacts with G proteins • Adrenergic receptors are typical • Note desensitization by phosphorylationby protein kinase A

  4. 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

  5. cAMP and Glycogen Phosphorylase Earl Sutherland discovers the first second messenger • In the early 1960s, Earl Sutherland showed that the stimulation of glycogen phosphorylase by epinephrine involved cyclic adenosine-3',5'-monophosphate • He called cAMP a "second messenger" • cAMP is synthesized by adenylyl cyclase and degraded by phosphodiesterase

  6. Signalling Roles for G() A partial list • Potassium channel proteins • Phospholipase A2 • Yeast mating protein kinase Ste20 • Adenylyl cyclase • Phospholipase C • Calcium channels • Receptor kinases

  7. Stimulatory and Inhibitory G G proteins may either stimulate or inhibit an effector. • In the case of adenylyl cyclase, the stimulatory G protein is known as Gs and the inhibitory G protein is known as Gi • Gi may act either by the Gi subunit binding to AC or by the Gi complex complexing all the Gi and preventing it from binding to AC

  8. Figure 19-13 Activation/deactivation cycle for hormonally stimulated AC. Page 674

  9. Figure 19-16 Mechanism of receptor-mediated activation/ inhibition of AC. Page 676

  10. 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

  11. Phospholipase targets

  12. Phosphotidyl inositols as secondary messengers

  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. Many different activators of phospholipase

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

  16. Protein kinase C: integration 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 • Phosphorylates Ser,Thr • Phorbol esters are apparent analogues of DAG • Signals terminated/modulated by cellular phosphatases dephosphorylate target proteins

  17. 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

  18. 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

  19. Patch clamp

  20. 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

  21. Calmodulin

  22. 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

  23. MAP Kinase Cascade

  24. Single transmembrane receptors • Receptor tyrosine kinases • Dimerization and cross-phosphorylation • Insulin receptor • Epidermal growth factor receptor

  25. Insulin receptor binding of peptide causes dimerization and cross- phosphorylation on Tyr residues

  26. Insulin signaling • Binding  Dimerization  Phosphorylation  • Binding and phosphorylation of IRS1,2  • Binding and activation of PI-3-K by IRS  • Formation of PIP3  • Activation of PIP3 dependent protein kinase and kinase cascade  Ca2+ release, activation of glycogen synthase kinase, etc.

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

  28. EGF signaling • Binding of 2 growth factor peptides • Dimerization • Phosphorylation of Tyr residues at C-terminus • Binding of adaptor protein Grb2 (SH2 – binds phosphotyrosine) • Recruits Sos (SH3 – binds proline rich region) • Ras G-protein binding and nucleotide exchange • Activation of MEK  Activation of ERK  transcription, etc. • Termination by phosphatases and ras GTPase activity

  29. When signal molecule misbehave

  30. 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.

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

  32. 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

  33. 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