3. Signalling

Learning Outcomes

Evaluate how regulation is accomplished through intracellular signalling Understand some important principles of signalling: modularity, feedback, use of second messengers, signal amplification and their application Define how signalling pathways can modulate important physiological functions, and how these are involved in diseases Describe the major constituents of the most important signalling cascades and their mode of action Describe the integration of signals into networks, cross-talk between different signalling cascades  

GPCRs (G-Protein Coupled Receptor)

2 major types of cell surface receptors initiate signalling cascades:  GPCRs and Enzyme-linked receptors Both initiate kinase cascades and second messengers  Extracellular ligands for these receptors are very diverse GPCRs (also called seven transmembrane, 7TM, receptors)  Examples of ligands: light, sugars and derivatives, nucleotides, small proteins Affect every level of human physiology: hormone secretion, exocytosis, neurotransmission, development, viral infection, smell, taste, vision Smell, taste, vision extracellular stimuli are exclusive to these receptors unlike other signals

Example of GPCR= B-adrenergic receptor

Conformational flexibility of receptor changes upond ligand binding  Ligand (adrenaline)  binding closes space at extracellular side between TM 3,5,6 and forces TMs apart at the cytosolic side  This induces a conformational change in 5/6 loop promoting signal transduction: 6 moves away from 3 and closer to 5 to allowing coupling of 5/6 to ligand

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Trimeric G-proteins

G proteins are grouped according to their downstream targets or a specific type of GPCR they are activated by, subscript next to alpha (a) describes: s (stimulatory)-             activated by adenylyl cyclase  (main class) i (inhibitory)-                  inhibits adenylyl cyclase  o, q-                                      activate the PLC/Ca 2+ pathway olf (olfactory) -              coupled to smell receptors t (transduction)-          coupled to rhodopsin in the eye G proteins/GTPases different classes do different things and the output of cell signalling depends on which proteins are present

G-protein cycle

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G-protein eg Ras or trimeric G-protein Inactive: Binds tightly to their nucleotide cofactor GDP Need a GEF(guanine nucleotide exchange factor) to release GDP to allow exchange GTP can then spontaneously bind due to its higher concentration than GDP in the cell  Active: G-proteins are GTPases but have low activity  Need a GAP (GTPase activating protein)  to help hydrolyse GTP, leave GDP remaining GAPs are specific to different GTPases

Signal handover

G proteins make a transient protein complex with GPCR then effector  For a trimeric G-protein: GEF=GPCR Activated GPCR (GEF) binds to G-protein and exchanges GDP for GTP in  Gα subunit , G-protein falls apart into separate subunits Gα binds downstream protein, adenylyl cylase (GAP) Upon GTP hydrolysis, transient Gα-adenylyl cyclase interaction is lost :  negative feedback - only activate signal as long as GPCR is activated by stimulus On membrane surface: GPCR is TM protein, Gα has lipid anchor which anchors it to the mem, AC is TM helices -allows proteins to find each other easily

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GPCR signalling through adenylyl cyclase

Some GPCRs activate AC in adipose tissues and some inhibit it ​​​​​​​The balance of stimulatory and inhibitory inputs determine the physiological response of the cell Stimulatory hormones: Adrenaline, Glucagon, ATCH Inhibitory hormones: PG, Adenosine

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GPCR decides how a signal is interpreted

The same signal can exert different effects in specific cells by signalling  through a different GPCR Allows target cells to activate physiological responses that are appropriate for a given tissue But at the same time utilise common signalling components

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Making a chimeric GPCR: Experiment

Proving where G-protein interacts (couples with) GPCR  Take 2 receptors that both respond to adrenaline (last slide) but bind different Gα-proteins either stimulate or inhibit AC Cut receptors in half, recombine and view outputs: Using 5, 6, 7 TM from stimulatory receptor results in stimulation  Using 5, 6, 7 TM from inhibitory receptor results in inhibition  Proves that G-protein is coupled to regions of helices 5/6/7  (5/6 cytoplasmic loop C terminus)

Second Messengers

Small, non-protein, intracellular molecules that are produced/released upon activation of a signal  Can themselves activate downstream targets- amplify a signalling cascade Their localised production in the cell and constant destruction  ensures a localised target response  Examples: DAG, cAMP, IP3, cGMP cAMP discovery: Took liver (glycogen stores) and fractionated: membrane contains GPCRs, G-protein, AC and cytosol contains glycogen phosphorylase Add adrenaline to membrane: membrane produces cAMP but no glycogen phosphorylase present to activate  Remove membrane fraction leaving behind a solution containing cAMP Mix cAMP solution with cytosol (no GPCR, no AC): activation of glycogen phosphorylase Proves that membrane produces a small molecule that transfers signal from membrane to glycogen phosphorylase

Targets of cAMP signalling: Protein Kinase A

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cAMP binds regulatory subunits of PKA Thus, activating PKA by releasing the active catalytic subunits Catalytic subunits can phosphorylate targets:  nuclear receptors, CREB (cAMP responsive element binding protein), Ca2+ channel in the ER and others There are 3  isoforms of catalytic subunits and 4 isoforms of regulatory subunits Various cellular responses to cAMP  in various tissues, most responses are driven by adrenaline (fight or flight)

Glycogen Metabolism

Glycogen Breakdown: Glycogen phosphorylase (GP) = cleaves off glucose  Glycogen Synthesis: Glycogen synthase (GS) = adds glucose units  Glycogen synthesis uses UDP-glucose (high energised form of gluocse) and glycogen Release of UDP gives energy to the system  Generateon of UDP glucose needs more energy than what you get from release of glucose from glycogen so: Either have to synthesise OR breakdown glycogen but NOT both together Tightly coupled to only have one active at a time

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Effects of cAMP on glycogen degradation

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PKA activation leads to stimulation of glycogen breakdown Activation of GP kinase: phosphorylate and activate GP including glycogen breakdown Inactivation of protein phpsphatase (PP) that would otherwise inactivate GPK and GP- PKA phosphorylates a small inhibitory protein that inhibits PP  PKA inactivation leads to inhibition of glycogen breakdown Activation of PP that dephosphorylates and inactivates GPK and GP

Effects of cAMP of glycogen synthesis

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PKA activation leads to inhibition of glycogen synthesis: Inactivation of GS by phosphorylating it  PKA inactivation leads to stimulation of glycogen synthesis: Activation of PP that dephosphorylates and activates GS Simultaneous activation of synthesis and inhibition of degradation or other way round - tight control of selected metabolic step  

Another second messenger: Ca2+

Regulation of Ca2+ levels in the cytosol: Ca2+ channels in the plasma membrane (CRAC, P2X) and the ER (IP3R) when activated provide a local increase in cytosolic Ca2+ (only transient increase) The ER Ca2+ pump ATPase (SERCA) constantly removes excess cytosolic Ca2+ into the ER (maintain low conc in cytosol compared to outside cell/inside organelles) Cytosolic resting [Ca2+] is 10,000 fold lower than concentration in the ER or outside the cell, active Ca2+ only requires a 10 fold increase of this

Calcium signalling

Examples of calcium signalling Muscle contraction: calcium exposes myosin binding sites, myosin binds and promotes actin movement and muscle contraction Neurotransmitter release: local calcium influx into synapse, promotes fusion of neurotransmitter containing vesicles to generate neurotransmitter response  

Calcium signalling: Calmodulin

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Specific example: Activation of Calmodulin Cytosolic calcium conc kept at a low steady state Various signals can trigger a local increase in calcium conc Local increase and binding of Ca2+ induces large conformational change, activating calmodulin Allosteric activation of calmodulin by Ca2+ allows calmodulin/Ca2+ to bind other targets such as kinases  Kinase (CaM-kinase II) is inactive until it binds Ca2+/calmodulin and then autophosphorylates itself and becomes fully active, can phosphorylate other targets

Summary

Evaluate how regulation is accomplished through intracellular signalling: Signalling involves transduction steps that can alter enzymatic activity: e.g. GP, the binding properties of an effector protein: e.g calmodulin, or the assembly of a protein complex: e.g. trimeric G-protein Understand some important principles of signalling: Second messengers are small molecules that are produced or released to transiently activate proteins. E.g. cAMP is generated downstream of adrenaline signalling and activates PKA. There are many different GPCRs, these couple to a variety of G-proteins,  different cells use this  for different functional outputs Define how signalling pathways can modulate important physiological functions, and how these are involved in diseases: Glycogen metabolism is regulated by adrenaline signalling (fight or flight response) Adrenaline can activate different types of responses dependent on the available downstream signalling components, and cAMP can have different functions in tissues depending on the targets available in each cell Describe the major constituents of signaling cascades and their mode of action: Second messengers are small molecules signal transducers. GPCRs are major cell surface receptors. G-proteins are molecular switches. Kinases (e.g. PKA) phosphorylate target proteins to alter their activities – e.g. GPK, GS, PP

Enzyme-linked receptors

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Ligands induce receptor dimerisation  Activates enzymatic activity of receptor on the cytosolic side through: Mutual trans-phosphorylation of the 2 subunits  (part of receptor structure) Recruitment of a catalytic subunit from the cytosol, which then becomes activated (recruited enzyme) Active enzyme phosphorylates and activates targets, initialling signalling cascade

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Major classes of enzyme-linked receptors

Receptor Tyrosine Kinases (RTKs)

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All have in common: intracellular kinase domain which is activated by receptor dimerisation Extracellular domains are very different but allow ligand to bind and dimerise

Phospholipases (PLC)

Activated by either a suitable Gα protein or an activated RTK (same downstream signalling) Is an enzyme so amplifies signal Generates second messengers from an important signalling lipid  Provides cross-talk between GPCR (environmental cues) and RTK (growth hormones)

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Enzymatic function of PLC

Activated  PLC hydrolyses PI(4,5)P2 (bisphosphate) -> diacylglycerol (DAG) and inositol triphosphate (IP3) Both DAG and IP3 act independently as second messengers PKC is a common downstream target of both DAG and IP3 IP3 releases Ca2+ from ER (another second messenger)

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About inositol (just for understanding)

Inositiol is a sugar derivative with 6 OHs, any other than 2' OH can be phosphorylated  When inositol forms a phospholipid, eg PI(4,5)P2, 1'OH always phosphorylated Hydrolysis of phospholipid produces triphosphate as the phosphate from glycerol in phospholipid remains attached to inositol 

Ca2+ release through IP3-gated channels

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Second messenger IP3 moves to the ER and activates IP3-gated Ca channel to release Ca2+ Ca2+ release is transient Increases Ca2+ concentration about 10- to 100-fold locally around the channel IP3 “degradation” is either by: phosphatases removing the 4’ or 5’ phosphate, or a kinase adding a 3’ phosphate

Example of Ca2+ signalling: PKC activation

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Protein kinase Cs are serine/threonine kinases that contain a regulatory domain attached to the catalytic domain, 3 subgroups: Conventional (α, β, γ) = activated by DAG and Ca2+, have kinase domain, calcium binding domain, glycerol binding domain Novel (δ, ε, η, θ) = activated by DAG only,  discovered by sequence (some homology) don’t need calcium but still similar substrate binding sites and targets Atypical (ζ) = activated by ceramide, even less like original PKC (conventional) differ in their activation but similar substrate binding sites and targets

Rubrica: : All PKCs are related by sequence but changes in their activation over evolution, the kinome is a diagram of all protein kinases and how they are related

Diverse examples of PKC targets

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Targets phosphorylated by PKCs modulate: Metabolism (GSK3β- glycogen synthase kinase 3β) Other signalling pathways (MAP kinase) Cytoskeleton, movement (CPI-17 inhibits a myosin phosphatase) Different PKCs also have diverse cellular effects in activating/ inhibiting eg: migration, autoimmunit,adhesion, apoptosis etc  

RTK signalling (receptor tyrosine kinases)

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Activation of RTKs: Ligand binding sites on both receptor halves, form homodimer After dimerisarion get transphosphorylation on intracellular side : each  lip  phosphorylates and activates other lip tyrosine Receptor is activated and phosphorylation of additional tyrosine residues 

RTK signalling involves a lot of domains

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Activated insulin receptor is a platform for intracellular signal transducing molecules  Insulin receptor substrate -1 (IRS-1) binds receptor (could bind other molecules eg PLC that all share ability to bind phospho tyrosine) Phosphorylated IRS-1  provides docking site for other signalling components that may be inactive individually  Another point of regulation- phosphatases can dephosphorylate to stop signal transdcution Modules bind the receptor via a phosphotyrosine (pTyr) 

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2 types of modules that bind pTyr = PTB and SH2 domain Specificity- only need a pTyr for binding how do you ensure that the recruited signalling module is specific for the signal Each SH2 domain binds to a distinct sequence of amino acids surrounding the pTyr: Shc adaptor protein needs a Leu or Val at 3rd position after pTyr Lck kinase needs Ile at 3rd position after pTyr PLC-γ1 phospholipase needs hydrophobic residue at 2nd position after pTyr Syp phosphatase needs specific residues upto 5 aa away from pTyr Specific and associated with a switch (only activated if  tyr is phosphorylated)

Structure of SH2 domain

IRS-1 PTB domain binding specificity

The phosphotyrosine binding domain (PTB) of IRS-1 specifically binds to pTyr of the insulin receptor (IR) and interleukin-4 receptor (IL-4R) Specificity of binding is determined by the aa preceding the Tyr: Alanine (in IL-4R) is the best  Glutamate in IR reduces affinity 50-fold- changing glutamate in IR to alainine converts affinity of IR to that of IL-4R Aspartate and glutamine in EGF receptor or neurotrophin receptor(TrkA) shows very weak to no binding     

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Phosphoinositide kinase mediated signalling

Phosphoinositol-3-kinase (PI3K) is one of the signalling molecules recruited by IRS-1 p85 subunit of PI3K contains a SH2 domain that binds to phopshorylated IRS-1 p110 catalytic subunit of PI3K phosphorylates PI4P or PI(4,5)P2 at the 3-position  This adds another phosphate to inositol head group and creates a binding site for yet other domains  (like the PH domain)

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Phosphoinositide kinase mediated signalling

PI (3,4)P2 (made from phosphorylation at 3-position of PI4P ) provides a binding site for PH domain of PKB (protein kinase B) Binding of PH domain onto the 3-phosphate of phosphoinositols= partial activation of the PKB and is now localised at the plasma membrane  PKD1 (activator of PKB) also contains a PH domain so is recruited to same site as PKB  Close proximity of PKB and PKD1 causes phosphorylation of the activation lip by PKD1= fully activated PKB      

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Targets of PKB

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Rubrica: : Insulin wants to get rid of glucose, PI3 is recruited to IRS activated by insulin receptor, main downstream target of insulin receptor is PKB

Kinase mTOR: central regulator of growth

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TOR: target of rapamycin (m stands for mammalian, discovered using yeast genetics) Influenced by(input from): Nutrients  Insulin signalling  through PKB Influences cell growth through: Protein synthesis  Autophagy(inhibits this) Respiration 

Kinase mTOR: central regulator of growthnase mTOR: central regulator of growth

mTOR is a critical gauge of metabolism which leads to its central role in physiological processes like aging and pathological ones like cancer Fasting inhibits mTOR and eating enhances mTOR In obesity mTOR is high mTOR activity linked to calorie intake and ultimately aging: restrict calories, inhibits mTOR, slow aging and healthier in old age  Rapamycin inhibits TOR which has a strong influence on growth, therefore used as a drug for: Immunosupression Cancer Longevity (in animals) Psychiatric conditions    

Summary

Evaluate how regulation is accomplished through intracellular signalling: A central node, such as PKC or mTOR can be activated by different stimuli and in turn regulates a host of cellular functions. Phosphorylation is an important switch that can activate/inactivate proteins or serve as a binding site to recruit signalling complexes. Understand some important principles of signalling: Specificity is achieved by fine-tuning the environment of common motives - e.g. SH2’s p-Tyr provides the central binding element, but specificity is provided by the surrounding amino acids. Signalling cascades are often modular – this allows the cell to use the same chemical tools, but diversify these through the fine-tuning of specificity. Define how signalling pathways can modulate important physiological functions, and how these are involved in diseases: mTOR is a central regulator of growth. It is regulated itself by nutrient/glucose levels. Insulin signalling promotes growth because it signals the availability of glucose=energy.  Describe the major constituents of signalling cascades and their mode of action: The second messengers DAG, IP3 and Ca2+ are linked and together activate PKCs. IR-PI3K-PKB-mTOR is an important metabolic regulatory cascade. Describe the integration of signals into networks:  PLC signalling can be initiated by either GPCRs (environmental cues) or RTKs (protein/peptide growth factors) / PKB activates PDE, which degrades cAMP / PKCs can activate MAPK pathway.

Compartmentalisation of signalling

Compartmentalisation in cytoplasm by putting receptors in endosomes Surface receptors ubiquitinylated following  prolonged stimulation  Monoubiquitination induces endocytosis (endosome provides second siganlling platform, properties different to PM Endocytosed receptors sequestered into internal vesicles of multivesicular body (MVB)- internal vesicle formed from invaginations of own membrane

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Compartmentalisation of signalling

Cellulat compartments differ based on the  phosphoinositide they have  Phosphoinositides can be interconverted by kinases and phosphatases This allows specific co-localisation of signalling components which results in their activation  

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Compartmentalisation to stop signalling

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Receptor activated when ligand is bound  Endocytosis puts receptor in endosome  If release if ligands not promoted by endocytosis (eg by change in pH of environment) receptor is still active and signalling  Active receptor delivered to MVB  Once in internal vesicle, receptors active cytosolic portion is sequestered from cytosol  (physical separation stops signalling) Receptors in MVB can be delivered to lysosome for destruction or recycled to PM

Phosphatases destroy domain binding sites

Many binding domains (SH2, PTB, PH) rely on phosphorylated binding sites When phosphate removed complex falls apart -> stops signal transduction Examples: PKA activates glycogen phosphorylase by phosphorylation, phosphatase removes phosphate and inactivates glycogen phosphorylase RTK generates large scaffold by phosphorylated tyrosine, phosphatases remove all phosphates, signalling platform falls apart(next slide) PKB: phosphatase cleaves lipid group from phosphoinositides and signalling platform falls apart (next slide)

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Phosphatases destroy domain binding sites

Desensitisation of GPCR signalling by kinase

Prolonged signalling can be due to tight binding between receptors and ligand or slow clearing  of ligand from blood  In such cases, negative feedback initiated by Gβϒ recruiting the G-protein-linked-receptor kinase (GRK) to the membrane: GRK phosphorylates receptor which promotes arrestin binding   Arrestin binding prevents trimeric G-protein binding  to GPCR (and promotes endocytosis of receptor) PKA or PKC can also phosphorylate GPCRs (negative feedback further downstream of pathway) 

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cAMP is degraded by phosphodiesterase

Further down signalling from GPCR, cAMP produced which activates PKA and amplifies signal  Signalling through adenylyl cyclase is antagonised by continuous breakdown of cAMP through phosphodiesterases (PDE)- ensures cAMP only acts when cell is stimulated  PDEs regulated themselves by PKA, PKB, calmodulin and others - fine tuning of cAMP response 

G-proteins have their own timer

G-proteins only active as long as GPCR is active  Upon GTP hydrolysis, activated Gα subunit becomes inactive Process slow for isolated Gα (several minutes), poor GTPase activity But speeded by GTPase activating proteins (GAPs) in the cell  Inactive Gα reforms timeric G-protein    

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G-proteins have their own timer

Only active as long as GPCR is active BUT pathogens can interfere and permanently activate this pathway by ADP ribosylation of G-proteins through bacterial toxins: Cholera toxin: Catalyses ADP-ribosylation of Gαs(stimulatory) = adds ADP-ribose from NAD+ to arginine residue at GTPase active site (NAD+ present in cell in large amounts because of respiration) Prevents GTP hydrolysis by Gαs - permanently active, constant cAMP production, constant PKA activation Pertussis toxin (whooping cough): Catalyses ADP-ribosylation at cysteine residue of inhibitory Gαi Incapable of exchanging GDP for GTP (GEF/GPRC cannot act on inhibitory Gα so cannot activate) Blocks inhibitory pathway- overproduction of cAMP and PKA    

Cross-talk between signalling pathways

Integration of GPCRs and RTKs pathways: Kinase targets (PKA, PKC, CaM-kinase, MAP kinase and PKB) have many common targets that need to be/ can be phosphorylated by more than one kinase Kinase targets such as PKA or PKC can phosphorylate, so regulate, members of other pathways  PLC is a common component of both GOCR and RTK signalling 

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Cross-talk at level of target proteins

At level of target proteins: Glycogen synthase is a target of both: PKB signalling originating at RTKs- insulin receptor pathway, inhibits glycogen synthase kinase, activates glycogen synthesis PKA signalling originating at GPCRs- Adrenaline activates cAMP pathway, activates PKA, inhibits glycogen synthase 

Cross-talk

At level of intracellular signal transducers: PKB: Inactivates IRS-1, providing negative feedback in its own pathway Inhibits cAMP pathway trhough PDE  Affects glycogen synthesis through GSK3 PKA: Feeds back on its own pathway (adenyl cyclase and PDE) as well as PLC, cAM kinase and Ca2+ levels through its effect on ER Ca2+ channels

Cross-talk

At level of receptors: PLC can be activated by both: Trimeric G-proteins coupled to GPCRs SH2 domain containing proteins that couple to RTKs

Pathways impacting on glycogen synthesis

Insulin and adrenaline cross talk: Insulin receptor activates PKB, inactivates glycogen synthase kinase (GSK3) Has effects on glycogen synthesis and protein synthesis: insulin counteracts effects of adrenaline through GSK3 whilst also activating protein synthesis 

Compartmentalisation streamlines crosstalk

Receptor activated at PM has different set of signalling molecules than if endocytosed because of different phosphoinositides marking different comparments By recruiting activated signal transducers and targets to endosomes, signalling can be channelled EEA1 and APPL are scaffolds that bind endosomal PIPs Endosomal recruitment of PKB promotes cell survival rather than growth- presents PKB with a different substrate  APPL binds PKB and GSK3 beta, PKB phosphorylates GSK3 beta and get self-survival APPL not present= PKB not recruited to endosome, remains in cytosol and phosphorylates TSC2 and activates mTOR- growth  Endosomal activation of PKB by MAPK pathway leads to over-growth EEA1 binds MAPK and PKB Activation of mTOR and overgrowth- hypertrophy  

Compartmentalisation streamlines crosstalk

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Different adrenaline signalling pathways

3 types of adrenergic receptors in the heart: 2 beta and 1 alpha receptor  Consequence of adrenergic signalling (through all 3 receptors) is muscle contraction through elevated Ca2+ levels and increase in heart rate Beta receptors elict stronger responses through PKA activation than alpha receptor through IP3 Prolonged activation leads to switching beta 2 receptor to inhibitory mode which leads to desensitisation of beta 1 receptor Fine-tuning of interplay between different receptor subtypes is important to allow increase in heart function when necessary and protection of heart against failure due to too much exercise

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Summary

Evaluate how regulation is accomplished through intracellular signalling: Growth, survival and hypertrophy can all be mediated by the same kinase – Akt – dependent on what other inputs the cell receives. Receptor downregulation is an important part of signalling regulation. Understand some important principles of signalling: Scaffolding and compartmentalization can direct the signalling to different outcomes. Signalling has to STOP – all signalling is transient. Define how signalling pathways can modulate important physiological functions, and how these are involved in diseases: Bacterial toxins can permanently activate signalling components, thereby defying the rule of transient signalling. Describe the major constituents of signalling cascades and their mode of action: Phosphatases can act to destroy signalling platforms. PIPs are intracellular postcodes for different organelles. Arrestins stop GPCR signalling by binding to the receptor to prevent G-protein coupling. Describe the integration of signals into networks: Signal transduction pathways do not act in isolation – cross-talk exists at all levels starting at receptors, through intermediate transducers down to the main kinases . Cross-talk is directed by common components as well as scaffolds and/or compartmentalization.