L5 Second Messenger Systems PDF

L5-second messengers Dr Andy Grant 5BBM0218 Physiology and Pharmacology of the CNS Secondary Messengers and Intracellular Signalling Dr Andy Grant [email protected] What is a second(ary) messenger? Primary messengers carry signals between cells (e.g. neurotransmitters, hormones) Secondary mes...

L5-second messengers Dr Andy Grant 5BBM0218 Physiology and Pharmacology of the CNS Secondary Messengers and Intracellular Signalling Dr Andy Grant [email protected] What is a second(ary) messenger? Primary messengers carry signals between cells (e.g. neurotransmitters, hormones) Secondary messengers carry signals within cells, following primary messenger signaling ↳ of target Mahe outering the activity the cells easier. Why are second messengers important? Regulate process signaling u Ne enesis Enzyme h wt ro In n o i t a m flam tg Ge n ou e s a e l e r r e t n t i o m i t s n p a r e t c o i r u c e N No Pro life rati on Synaptic plasticity e rit S yn a p t o g most on i s s e r e exp regulatio n Ce ia v ll y t i l bi Major themes Signal amplification Temporal localisation Sensitivity of response Selectivity of response small subregions from the cell Spatial localisation / Complexity vs. speed Loss of speed compared te action potential but much more complex. Consequences of signal localisation and amplification Together, mechanisms to amplify signals and also control their duration / location can produce very complex intracellular signals: slide ming Agonists produce different cellular responses by ↳ diggementsurface receptor subtypes. Acting - - Differentactions of ↳ Ach acts on muscle via calium to - 2a mones on Activating the vascular my receptors and have opposite receptors messenger pathways. increase intracellular effects. that ↓ receptor/second messenger. endothelial cells/smooth same couple to multiple Calcium induced calcium = releas calcium wave second Video by Steele, Yang, Taggart, Sweeney at https://www.youtube.com/watch?v=7LyZkNeyw9s Main types of second messengers Cyclic nucleotides: 3’,5’-cyclic adenosine monophosphate (cAMP) 3’,5’-cyclic guanosine monophosphate (cGMP) Molecules derived from lipid bilayers: Inositol 1,4,5-trisphosphate (IP3) Diacylglycerol (DAG) Arachidonic acid Gases: most frequent I Nitric oxide (NO) Carbon monoxide (CO) Ions: Calcium (Ca2+) Second messengers are usually produced by effector enzymes Effector enzyme Substrate Product Adenylate cyclase Adenosine-5’-triphosphate (ATP) cAMP Guanylate cyclase Guanosine-5’-triphosphate (GTP) cGMP Phospholipase A2 Phosphatidylcholine Phosphatidylethanolamine Arachidonic acid Phospholipase C Phosphatidylinositol 4,5bisphosphate (PIP2) IP3 and DAG Nitric oxide synthase L-arginine NO Exception: Ca2+ Release - into the or removal cytoplasm Not created or of destroyed) Ca Second messenger activity is associated with all 3 classes of transmembrane receptor 1. G-protein coupled receptors (GPCRs) Gas - ­ cAMP production by adenylate cyclase stimulatory Gai - ¯ cAMP production by adenylate cyclase Inhibitory Gaq/11 - ­ IP3 and DAG production by PLCb 2. Ligand-gated ion channels Ca2+ entry 3. Tyrosine kinase-linked receptors Phosphorylation cascades activate effector enzymes (e.g. PLCg) and protein kinases ↳ Activates other second messengers e.g. IP3 How do second messengers modulate cellular activity in the nervous system? Rapid turnover of second messenger molecules – transient effects (secs) Longer-term responses require covalent modification of messenger proteins E.g. memory. Changes larger -> persist than second concentration Achieved by phosphorylation of target proteins (e.g. ion channels, transcription factors, enzymes) by protein kinases Effects reversed by dephosphorylation of target proteins by phosphatases Modulating protein activity: changing shapes structure determined by charge distribution -> changes shape :. changes function. structure function Serine Threonine Tyrosine Types of protein kinase Initial estimates were for 1-2,000 protein kinases Sequence analysis of human genome suggests 518: Serine/Threonine kinases – e.g. PKC, PKA, PKG, MAPK, CaMK Receptor Serine/Threonine kinases – e.g. TGFb receptor Dual specificity protein kinases – Tyrosine/Threonine – e.g. MEKs Receptor tyrosine kinases – e.g. neurotrophin receptors Non-receptor tyrosine kinases – e.g. Src family ~400 Serine/threonine and dual specificity kinases ~100 Tyrosine kinases identified to date Also ~ 50 lipid kinases – e.g. PI-3-kinase Signal amplification: NGF, protein kinase cascades and pain sensitisation What is signal amplification? Dopamine GPCR AC = Adenylate Cyclase D1 receptor a Gas g serise b patway cAMP AC cAMP cAMP cAMP PKA cAMP PKA PKA cAMP cAMP PKA cAMP Activation of one receptor by a single extracellular transmitter molecule produces synthesis of multiple second messengers of effect and alters the activity of multiple targets size inmeases NGF sensitises pain pathways: amplification by protein kinase cascades Nerve Growth Factor (NGF) is synthesised by many different issues organ type cell types and stimulates organ innervation Drives -> Key role in driving neuronal proliferation and survival during nervous system development m adults: Released during tissue damage and inflammation from epithelial cells, mast cells etc. and increases excitability of sensory nerves. Binds to trkA receptors on peripheral neurons Altered pain processing through: - Altered gene expression (e.g. CGRP, substance P) - Reducing NaV activation threshold; TRPV1 receptor activation and trafficking to the cell surface Potential to treat chronic pain by blocking NGF signalling Dimerisation of NGF receptors Nerve growth factor (NGF) signals via trkA receptors Dimerisation to became active Autophosphorylation Inactive monomers Active dimer Active trkA receptors couple to GRB2 and Sos Active trkA dimer Ras Phosphates sites binding GRB2 = Growth factor receptor-bound protein 2, an adaptor protein Sos = Son of sevenless, a guanine nucleotideexchange factor ↳ Binding size our Ras Mas trkA/ GRB2 / Sos complex activates Ras Ras Ras (from RAt Sarcoma) = small GTPase, active when bound to GTP GTD achclede Ras activates MAP kinase cascade Ras-> Raf -> MER -> MAPK signal amplification Raf = MEK kinase, a protein kinase MEK = MAPK / ERK Kinase changes gene a a = MAPK = Mitogen-Activated Protein Kinase (ERK1/2) expression neurons different proteins produced. trkA receptor signal cascades in neurons trkA scaffold far Amplification many Amplificationusing within one cascades cascade other kinases Extracellular Intracellular Activates many ↑ 512 Y-P GRB2 + Sos +++ Ras Raf-1 PI-3-K PLCg PKC 706 707 Y-P Y-P 751 785 Y-P Y-P ++ MEK + +++ ++ ERK1/2 ++ ++ Cytosolic Targets Gene Expression TRPV1 channels (CREB) channels CREB = cAMPTRPV1 Response Element-Binding & ⑨ Signal amplification: key points Signal amplification produced by increasing the number of active signaling molecules at each stage in a cascade, and by using multiple pathways: Few primary messenger molecules need to reach target cell Þ sensitive and efficient A single primary messenger can produce very diverse cellular responses ….but why doesn’t the cell always have all its signaling pathways switched on? ↓in space time + Spatio-temporal localisation: cAMP, scaffolds and memory - Local [cAMP] influences memory formation cAMP, via activation of PKA and CREB phosphorylation, modulates memory formation Short term effects by phosphorylation and increased trafficking of glutamate receptors, Kv channels and GABA receptors Long term effects through transcriptional regulation via knockout CREB formed. CREB ~ mice a = no memories CREB knockout mice have memory deficits, and impaired ↑ patterns of neuronal hippocampal LTP firing strengthens synapse. = [CREB suggested to be important survival factor for dopaminergic neurons in the substantia nigra] cAMP synthesis and metabolism AC = Adenylate Cyclase AC PDE = PhosphoDiEsterase mormaoxoxcomacology or I CAMPaRed ATP be multiple can cAMP AMP Temporal Localisation isneals pat, YePDE IIImavating 8 isoforms of adenylate cyclase in the CNS Bound to the plasma membrane (TM domains) All activated by Gas and inhibited by Gai Isoforms I, III and VIII activated by calcium / calmodulin Isoforms V and VI inhibited by calcium (CaM-independent) close to receptors. Effects of cAMP are mediated by PKA cAMP cAMP BindingafcAMP came a to dissociate, I to act on R R C C Protein Kinase A (PKA) Heteroetramer units cAMPcAMP anang targets ↑ phosphorylationR ATP + C R C ATP ADP + Ser-P / Thr-P Targets: Ion channels (e.g. glutamate receptors) Enzymes (inc. phosphodiesterase) Transcription factors (e.g. CREB) AKAPs produce intracellular cAMP domains AKAP = A-Kinase Anchoring Protein GPCR a microdomains g cAMP PDE PKA AMPor turned quickly AMPA / NMDA Limits Kv channels rspace ancheringGABA receptors w AC cAMP PKA PDE AKAP 4 again signal augy time CREB Transcriptional effects cells & Gas b in AMP Spatio-temporal localisation: key points Using scaffold proteins to restrict secondary messenger production to subcellular regions maintains specificity of effects AKAPs also bind other signaling molecules, such as PKCs and protein phosphatases to integrate different signal together signalling pathways pathways Bring Physical properties of second messengers (e.g. DAG) ↓ Large also restrict signalling to specific regions + molecule lipophilic ↓ Localized to inner leagretof plasma membrane Spatio-temporal localisation: Avoiding Ca2+ excitotoxicity 100 co Ca2+ is an essential second messenger… E.g. but highly toxic to cells regulator of muscle contraction Ca2+ ions regulate great diversity of processes (e.g. memory formation, vesicular trafficking, neurogenesis) Most target proteins don’t bind Ca2+ ions directly – effects via calmodulin (CALcium MODULated proteIN; CaM) Many effects also rely on activation of Ca2+/calmodulindependent protein kinases (CaMKs) Serine / threonine kinases Two types: - Specialised (e.g. Myosin light chain kinase) - Multifunctional (e.g. CaMKII, primarily expressed in ↓ neurons) multiple targets One target of CaMKII is CREB But…. Ca2+ is an essential second messenger… but highly toxic to cells Sustained increases in [Ca2+]i cause apoptotic / necrotic cell receptors death – excitotoxicity permeability ah a can Often due to increased glutamate and NMDA receptor activity and / or decreased cytoplasmic Ca2+ buffering Elevated global cytoplasmic Ca2+ activates proteases, phospholipases and endonucleases, damaging cytoskeleton, cell membranes and DNA ↳Destroys membrane, DNAetc. Associated with pathology of stroke, traumatic brain injury and neurodegenerative diseases (e.g. Parkinson’s Disease, Alzheimer’s Disease) How does the cell regulate [Ca2+]i? [Ca2+] [Na+] 2mM 145mM 1 20,000 1 9.7 vsteep gradient due to excitotoxicity. 0.1µM 15mM Not free calcium ions aytoplasm!!! in Provens thatbind wont signalling effect. 9 NCKX = Sodium / Calcium / Potassium Exchanger SERCA = Smooth Endoplasmic Reticulum Calcium ATPase from Clapham (2007). Cell ⑤ How does the cell regulate [Ca2+]i? [Ca2+] 2mM 1 2,000 L on 7 = calcium induced calcium releas signal I ~1µM M Intracellular stones V ↳ in tightly Calcium ER localised waves from Clapham (2007). Cell Spatio-temporal localisation: key points In addition to the site of production / release, the rate of second messenger breakdown / removal is also important Stopping the activity of second messengers quickly prevents unwanted signaling effects Rapid on-off signals are more energetically efficient, and provide greater temporal resolution Learning Objectives After this lecture, with some further reading, you should be able to: Understand the fundamental role of 2nd messengers in modulating cellular activity ü Describe the association of 3 major receptor classes with 2nd messenger function and its role in signal amplification ü Understand the importance of spatial and temporal localisation of 2nd messengers to signal specificity ü Give examples of multiple effector enzymes that enable different classes of receptor to couple to the same response ü Give examples of different 2nd messengers that converge on a common target ü References Standard course textbooks Carnegie, G.K., Means, C.K. & Scott, J.D. (2009) A-kinase anchoring proteins: from protein complexes to physiology and disease. IUBMB Life 61(4), 394-406 Clapham, D. (2007) Calcium signaling. Cell 131, 1047-1058 Rizzuto, R. & Pozzan, T. (2006) Microdomains of Intracellular Ca2+: Molecular Determinants and Functional Consequences. Physiological Reviews 86, 369-408 Barker, P.A., Mantyh, P., Arendt-Nielsen, L., Viktrup, L. and Tive, L. (2020) Nerve growth factor signalling and its contribution to pain. Journal of Pain Research 13, 1223-1241