Cellular Signal Transduction-2

Cellular Signal Transduction Part 1: Signal molecules andtheir recognition (2)Important!•Only a few types of cells can directly response to external signals.•Most signals come inside of an organism to enable the communication between cells.•Biological reactions are the final readout of signal transduction.Internal signal recognition Example 2: Hormone actionWhat happens after exercise? •Energy consumption leads tothe decrease of blood glucose level.•Stimulated adrenalin secretion results in glycogenolysis, lipolysis, enhanced heartmuscle contraction andincreased blood pressure, etc.Where to start with?Some fundamental observations •Phosphorylase played a rate-limiting role in the conversion of glycogen to glucose.•Hormones stimulated the activity of this enzyme.•Glycogen synthase activity decreased while phosphorylase activity increased. What was the nature of phosphorylase activation?•Molecular weight didn't change much during the inactivation, the loss of a phosphate group could be a possibility.Ultracentrifugation of LPand inactive LP showedthat they had the samesedimentation constant.Phosphatase inactivated the enzyme!What was the relation between phosphorylase& hormones?A demonstration in a cell-free system.ATP and Mg2+requiredRall T. et al. (1957). JBC 224:463-475•Hormones stimulated the activity of this enzyme.•Hormones responses were lost when cellular debris was removed after centrifugation.•They could be restored by recombining the particular fraction with the original supernatant.The relation between phosphorylase &hormones•A heat-stable factor was produced when incubating the hormones with the particular fraction. •Hormones increasedthe accumulation ofcAMP in liverhomogenates bystimulatingadenylate cyclase.•Phosphodiesterasecatalyzesbreakdown of cAMP.Phosphodiesterase (PDE) keeps the balance.Location: cell membrane (except mature red blood cells).Adenylate cyclase (AC):ATPcAMP + PPi5′-AMPACPDEcAMP: a second messenger Transmembrane signallingCrucial observations!•Hormones could not activate the enzyme unless guanosine triphosphate(GTP) was also present.Rodbell M. et al. (1971). JBC 246, 1877-1882.•GTPase activity was associated with the activation of AC.Cassel D. et al. (1976). Biochem. Biophys. Acta452, 538-551.•Nonhydrolyzable analogue of GTP could activate AC without the need for hormones.Londos C. et al. (1974). PNAS USA 71, 3087-3090.•Mutant S49 cells(UNC1) hadnormal receptorsand AC but failedto generate cAMPsignal in responseto appropriatehormones, butcould bestimulated byGpp(NH)p.Haga T. et al. (1977). PNAS USA 74, 2016-2020.•Two proteins were required:–Adenlate cyclase, present in cyc-membrane;–A stimulatory protein, deficient in cyc-membrane.The role of hormone receptor was to regulate the interaction between these two components.Gαmutation in this cell line.G Protein Coupled Receptors, GPCRS Sdomain)SG protein1994 Nobel prize was awarded to Gilman A. & Rodbell M. for their discovery of G protein.Glycogenolysis ↑RepinephrinecAMPG protein GGilman A Rodbell MHypothalamus &pituitary glandlungsHeartliver Adrenals G proteins respond to hormonesG proteins make senseSignal molecules •Extracellular / intercellular signal molecules: first messengers •Intracellular signal molecules: second messengersFirst messengers Produced by cells to signal to other cells •Hormones•Neurotransmitters and neuropeptides •Growth factors•Cytokines•Vasoactive agents,•Gas molecules•Adhesion moleculesMediation of signals •Signals can act at short or long-distance.•Signalling by extracellular molecules can be classified into 6types of signalling:–Endocrine signalling–Synaptic signalling–Paracrine signalling–Autocrine signalling–Contact dependent signalling–Gas signallingCarlsson A. discovered that dopamine is a neurotransmitter. Shared with Kadel& Greegardfor 2000 Nobel PrizeParacrine signalsLocal mediator, e.g.,HistamineGrowth FactorsCytokinesOnly affect target cells in close proximity to secreting cells.Akdis CA, et. al. Eur. J, Pharmacol. 533 (2006) 69–76.Autocrine signals Growth factors e.g,EGF , FGFPDGF, NGFCells respond tosubstancesproduced andreleased by thesame cellsDiscovery of EGF•EGF accelerates corneal re-epithelialization in rabbits with wounded corneas.•a 53-residue polypeptide, entirely devoid of alanyl, phenylalanyl, or lysyl residues, and with 3 internal disulphide bonds.•EGF stimulates protein kinase phosphorylation.•EGF receptors are tyrosine kinases.1986 Nobel prize was awarded to Stanley Cohen&Rita Levi-Montalcini for their discoveries ofgrowth factors.Direct cell contact dependent.About Notch•Notch is a large, single transmembrane protein that acts as a receptor for the ligands: Serrate (Jagged) and Delta.Adopted from Allman et al. (2002) Cell. 109, S1-S11Activation of Notch signallingGas signals(also serve as second messengers)nitric oxide synthaseL-arginine Nitric Oxide (NO)NO binds to and activates soluble guanylate cyclase.Discovery of biological effects of NO Some observations:•cGMP as a second messenger involves in many biological functions.•Nitrite stimulates guanylate cyclase activity. •Nitroglycerin can dilate blood vessels and relax vascular smooth muscle cells.•Nitroglycerin releases NO.Is NO an active intermediate or proximal activator of guanylate cyclase?Rat lung tissueKatsuki S et al., (1977). J Cyclic Nucleotide Res.3,23-35Bovine tracheal muscle tissueKatsuki S et al., (1977). J Cyclic Nucleotide Res.3,23-35Murad’s lecture 1998FurchgottEDRF=NO, IgnarroCO is also a signal moleculeHeme oxygenasesExtracellular matrix: immobilized signal molecules•Collagens•Fibronectin, FN•Laminin, LN•Elastin•Proteoglycans•GlycosaminoglycansWhat are common in extracellular signal molecules?Extracellularmolecules activate signalling pathways to effect different cellular responses.Intermediates Variable responses。