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Signalling at Cell Surface

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  1. Signalling at Cell Surface 2 April 2007

  2. Receptors

  3. Classification of receptors • Intracellular receptors (for lipid soluble messengers) • function in the nucleus as transcription factors to alter the rate of transcription of particular genes. • Plasma membrane receptors (for lipid insoluble messengers) • Receptors function as ion channels • receptors function as enzymes or are closely associated with cytoplasmic enzymes • receptors that activate G proteins which in turn act upon effector proteins, either ion channels or enzymes, in the plasma membrane.

  4. Cell Surface Receptors • May work both fast and slow • Always use “second messengers”

  5. Cell-Surface Receptors Belong to Four Major Classes • G proteincoupled receptors : epinephrine, serotonin, and glucagon. • Ion-channel receptors: acetylcholine receptor at the nerve-muscle junction. • Tyrosine kinase linked receptors: cytokines, interferons, and human growth factor. • Receptors with intrinsic enzymatic activity

  6. Four classes of ligand-triggered cell-surface receptors

  7. RECEPTORION CHANNELS • multi-subunit, transmembrane protein complexes • complex is both the receptor and ion channel • stimuli: chemical, stretch or voltage • stimulus induces conformational change to open or close ion channel • two types: 1) ligand-gated ion channel 2) voltage-gated ion channel

  8. LIGAND-GATED ION CHANNELS • chemical stimuli bind to receptor and open or close ion channel • stimuli can be extracellular or intracellular • EXTRACELLULAR STIMULI: (neurotransmitters) • e.g. acetylcholine, dopamine, GABA, glutamate • INTRACELLULAR STIMULI: (second messengers) • e.g. IP3, cAMP, cGMP, Ca2+

  9. LIGAND-GATED ION CHANNEL AT THE SYNAPSE • occurs at gap (synaspe) between nerve and target cell • acetylcholine (ACh) released into synapse • ACh binds to ion channel on target cell, opens channel, influx of Na+ • enzyme acetylcholinesterase released into synapse to breakdown ACh

  10. ACETYLCHOLINE ANTAGONISTS • very potent neurotoxins • bind to receptor and prevent opening of Na+ channel • e.g. cobratoxin from Indian cobra • atropine from deadly nightshade • S. American arrow poison (curare) - very fast acting so shot animals don’t run too far

  11. VOLTAGE GATED ION CHANNELS • ion channel undergoes conformational change folllowing electrical stimulus • this “depolarization” opens the channel • leads to flow of Na+ into cell • constitutes an “action potential” • channel recloses

  12. Signaling pathways downstream from G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs)

  13. Structural formulas of four common intracellular second messengers.

  14. Intracellular proteins • Two groups of evolutionary conserved proteins function in signal transduction • 1. GTPase switch proteins • Conversion from GDP bound inactive state to GTP-bound active state is mediated by guanine nucleotide exchange factors (GEFs) • Intrinsic GTPase activity hydrolyzes bound GTP to GDP + Pi

  15. GTP hydrolysis is accelerated by GTPase accelerating protein (GAPs) • Two classes of GTPase switch proteins: • Trimeric (large) G proteins • Directly bind to receptors • Monomeric (small) G proteins • Linked to receptors via adapter proteins and GEFs

  16. Common intracellular signaling proteins

  17. 2. Protein kinases and phosphatases • Human genome encodes 500 PKs and 100 PPs • Two types of PK • Those that P* OH group on Tyr residue • Those that P* OH group on Ser or Thr residues • PK is activated • By other kinases • By direct binding to other proteins • By second messengers

  18. Regulation of signaling • External signal decreases • Degradation of second mesenger • Desensitization to prolonged signaling • Receptor endocytosis • Modulation of receptor activity • Phosphorylation • Binding to other proteins

  19. G Protein-Coupled Receptors • A very large family of receptors coupled to trimeric G proteins • Activate or inhibit adenylyl cyclase • All have seven membrane spanning region • Ligands include: • Hormones, neurotransmitters, light activated receptors (rhodopsins), thousands of odorant receptors

  20. GPCRs and G proteins are involved in the regulation of many important physiological functions

  21. Signal transducing G protein has 3 subunits • G, Gß and G • G is the GTPase switch protein and modulates the activity of an effector protein • Effector proteins are either membrane bound ion channels or enzymes generating second messengers

  22. GPCR-mediated dissociation of trimeric G proteins has been demonstarted in fluorescence energy transfer experiments

  23. The activation/deactivation cycle of G proteins Agonist-receptor complex     + GTP   GDP GTP GDP  Inactive effector Active effector    Pi   Active effector GDP GTP

  24. G proteins can be linked to: • adenylate cyclase • produces cyclic AMP (cAMP) • guanyl cyclase • produces cyclic GMP (cGMP) • phospholipase C • produces inositol trisphosphate (IP3) • and diacyl glycerol (DAG) • ion channels

  25. G-Protein-Activated Enzymes

  26. first messenger receptor transducer amplifier second messenger

  27. Activity of bg subunits • Activation of K+ Channels

  28. G-Protein-Activated Enzymes • Generate new molecules - “second messengers

  29. G proteins and cAMP

  30. cAMP vs PKA

  31. cAMP and gene transcription

  32. Epinephrine case • Mediates body’s response to stress, when all tissues need glucose and fatty acids to produce ATP • ß-adrenergic receptors • Heart muscle: contraction • Smooth muscle cells of intestine: relax • 2-adrenergic receptors • Smooth muscle cells of endothelium, skin, kidney and intestine: constrict

  33. ß1 and ß2 adrenergic receptors are coupled to stimulatory G protein (Gs) • Actvates adenylyl cyclase • 1 adrenergic receptor is coupled to inhibitory G protein (Gi) • Inhibits adenylyl cyclase • 2 adrenergic receptor is coupled to Gq that activates another effector enzyme

  34. Bacterial toxins • Vibrio cholera • Catalyzes chemical modification of Gs that prevents hydrolysis of GTP to GDP • Active state • Bordetella pertussis • Catalyzes chemical modification of Gi that prevents release of GDP • Inactive state

  35. Critical domain of GPCR resides in C3 loop according to chimeric receptor expression experiments