Biol220 Cell Signalling Cyclic AMP the classical secondary messenger

Transcription

1 Biol220 Cell Signalling Cyclic AMP the classical secondary messenger

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3 The classical secondary messenger model of intracellular signalling A cell surface receptor binds the signal molecule (the primary messenger ). Occupation of the receptor is transduced into an intracellular signal by activation of an effector enzyme. The enzyme makes a secondary messenger that initiates spread of signalling information away from the vicinity of the occupied receptor. The secondary messenger transfers information by interaction with target proteins that promote activation/inactivation of protein kinases and/or protein phosphatases. Changes in the phosphorylation state of the protein substrates (e.g. enzymes, transcription factors) for these signalsensitive protein kinases/phosphatases bring about changes in cellular activity.

4 Ligand Receptor Transducer Effector Enzyme Membrane Cell Interior Secondary Messenger ACTIVATION Primary Protein Kinase PHOSPHORYLATION ACTIVATION Target A Target B Target C Other Protein Kinase(s) PHOSPHORYLATION Target D Target E Target F Fig. 1 The classical secondary messenger signalling system The classical secondary messenger mechanism

5 The cyclic AMP secondary messenger system Adrenaline, acting on a hepatic -adrenergic receptor, leads to the activation of adenylyl cyclase with the consequent production of cyclic AMP.

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7 The role of G-proteins in cyclic AMP signalling Heterotrimeric ( ) guanine-nucleotide binding proteins (G proteins) couple seven-pass hormone receptors to the enzymes (e.g. adenylyl cyclase) in the plasma membrane responsible for the generation of secondary messengers (e.g. cyclic AMP). Occupation of the receptor causes activation of the G protein, which, in turn, regulates the activity of the plasma membrane enzyme.

8 The G protein cycle In the inactive state, the G protein has GDP bound to a single GDP-binding site. Following occupation of the receptor, the hormonereceptor complex binds to the G protein and triggers the release of GDP in exchange for GTP.

9 This causes the breakdown of the G protein complex into a GTP-containing -subunit and a subunit complex. This is the active state of the G protein and the subunit directly activates the enzyme responsible for secondary messenger synthesis. Deactivation of the G protein is achieved by the breakdown of GTP to GDP on the -subunit. This is catalyzed by the intrinsic GTPase activity of the -subunit. The GDP-containing -subunit then binds the subunits thereby reforming the original inactive complex.

10 The G-protein cycle.

11 Enzymic synthesis and degradation of cyclic AMP Cyclic AMP is formed by the cyclization of ATP. The 3'OH group of the ribose unit attacks the - phosphoryl group of ATP to form a phosphodiester bond, with the concomitant release of pyrophosphate. Cyclic AMP is hydrolyzed to 5'-AMP by specific phosphodiesterases (PDEs).

12 Cyclic AMP-dependent protein kinase (PK-A) This enzyme mediates all the known effects of cyclic AMP in animal cells. The enzyme is a tetramer. Two catalytic (C) subunits are complexed with two regulatory R subunits to give an overall R 2 C 2 structure. The R subunits possess two non-identical, high affinity binding sites for cyclic AMP. Occupation of first site leads to a conformational change and the exposure of a second binding site on each R subunit. Binding to the second site leads to the dissociation of the complex and the release of free catalytically active C subunits.

13 PK-A is able to phosphorylate a wide range of target proteins. PK-A phosphorylates and activates the enzyme phosphorylase kinase that, in turn, is responsible for phosphorylation and activation of glycogen phosphorylase. Following activation of PK-A, protein phosphatase 1 (PP-1) also becomes inactivated. PK-A catalytic subunit complexed with PKI(5-24) and ATP analogue.

14 Adrenaline -Receptor Membrane Gs Adenylate Cyclase AMP PDE cyclic AMP ACTIVATION Protein Kinase A PHOSPHORYLATION ACTIVATION Target A Target B Target C Phosphorylaae Kinase PHOSPHORYLATION Target D Glycogen Phosphorylase Target F Fig. 2 The cyclic AMP signalling system in liver The cyclic AMP secondary messenger system.

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17 Adrenaline Cyclic AMPdependent control of glycogen breakdown

18 Control of phosphorylase kinase activity by cyclic AMP and Ca 2+

19 adrenaline adrenaline Cyclic AMP-dependent control of glycogen breakdown: activation of glycogen phosphorylase and inhibition of glycogen synthase.

20 The role of protein phosphatases In muscle some protein phosphatase 1 (PP-1) is located, together with enzymes of glycogen metabolism, within glycogen particles. Glycogen-associated PP-1 is composed of a catalytic subunit and a glycogen binding regulatory subunit. Adrenaline causes (cyclic AMPdependent) phosphorylation of the regulatory subunit, whereby the catalytic subunit is released from the glycogen particle. The freed catalytic subunit is inactivated by the adrenalineactivated protein phosphatase inhibitor proteins.

21 CREB Transcription Factors CREB (cyclic AMP response element binding) - protein is a DNA binding activity that binds to promoters of cyclic AMPinducible genes and mediates their induction in response to activation of the cyclic AMP pathway. Following an increase in [cyclic AMP], free PK-A catalytic subunits can migrate to the nucleus and phosphorylate CREB. Ser 133 is phosphorylated resulting in a 20-fold increase in CREB transcriptional activity.

22 The many roles of cyclic AMP.

23 Summary The cyclic AMP signalling system is the classical secondary messenger pathway. Occupation of a receptor is transduced into adenylyl cyclasemediated intracellular cyclic AMP production by the G-protein cycle. Accumulated cyclic AMP is able to bind to, and activate, the cyclic AMP-dependent protein kinase. This enzyme increases the phosphorylation state of specific target proteins (e.g. enzymes, transcription factors). The actions of adenylyl cyclase and cyclic AMP-dependent protein kinase are opposed by phosphodiesterase enzymes and protein phosphatases. These enzymes attenuate the signalling activity of the cyclic AMP signalling pathway.