Erhand Hohenester - Cell Signalling Flashcards

Question

What factors are associated with a transient response and a more long lasting response?

Answer 1

Signalling systems – persistence of response can be different **Transient response** - rapid response and decay 1. Fast turnover of signal mediators 2. Negative feedback loops 3. Rapid Adaptation **Long-lasting (permanent) response** - often the case in developmental signals 1. Switch-like behavior 2. Positive feedback loops

Answer 2

Negative feedback may lead to dampening of the signal  reduces the amplitude But... Negative feedback tend to have a delays resulting in oscillating outputs Why? Takes time for the cell to dampen the signal due to noise in biological systems leading to weak negative feedback loop response which diminishes quickly Solution to this? If a robust biological oscillations are required, we need positive feedback loops to counteract the effect of the negative feedback - amplify the entire system

Answer 3

CDK oscillates with a period of 1 hour – Robust oscillations Background - Cell cycle activated by cyclin which activates a CDK – drives cell cycle progression **Core negative feedback loop:** 1. The CDK-cyclin complex phosphorylates the anaphase-promoting complex (APC) 2. Promotes Cdc20 binding 3. The APC-Cdc20 complex polyubiquitylates the cyclin, targeting it for destruction. **Core negative feedback loop is sharpened/counteracted by two positive feedback loops** Positive feedback loop 1: 1. The CDK-cyclin complex phosphorylates the kinase Wee1 2. Inactivating Wee1 3. Prevents phosphorylation of CDK-cyclin complex - preventing CDK-cyclin complex inhibition --\> Promoting phosphorylation of APC Positive feedback loop 2: 1. The CDK-cyclin complex phosphorylates the phosphatase Cdc25 2. Active Cdc25 dephosphorylates the CDK-cyclin complex, 3. Activating it (double positive loop) --\> Promoting phosphorylation of APC

Answer 4

Logic gates – apart from feedback loops we also have logic gates 1. A and B need to be present to lead to an output 2. Neither A and B can be present in order to achieve the output 3. Either A or B OR Both can lead to an output 4. XOR – either A or B but not Both leads to output

Answer 5

Sustained input detector – used so that only a **sustained input yields an output but not a transient input** The system consists of... 1. Fast branch - happens quickly straight to the gate molecule - alteration in protein activity 2. Slow branch - happens slowly - doesn't interact quickly with gate molecule --\> change in gene expression AND Gate --\> Means that both fast and slow branch products need to be present to lead to an output Hence.... - Only during sustained input do we get elevated levels from the fast and slow branch - During transient input the fast branch increases but decays quickly which is followed by slow branch expression - both of which are not aligned in time yielding no response

Answer 6

Mitogen signalling – signal molecule leading to cell division (correct output) For cell division - we need the signalling molecule to induce an sustained input 1. Signalling by a mitogen leads to activation of MAP Kinase (Mitogen activated protein) 2. MAPK phosphorylates TFs directly and indirectly – leads to Fos1 transcription and translation - Fos1 is TF that is associated with transcription of genes involved in cell cycle progression 3. Fos1 itself is unstable, so it needs to be phosphorylated itself by MAPK 4. Hence, upon Fos1 transaltion, the input signal needs to be present and active to stimulate MAPK activity so that Fos1 can be phosphorylated and stabilize - allowing it to act as an effector. **Slow Pathway** - Fos1 transcription/translation **Fast pathway** - Fos1 stabilization via MAPK Both of the processed need to be present/aligned in time in order to obtain the desired output of cell division. Note - Feedback loops in oscillating signals require the mediator to have an intrinsic half-life/stability. Otherwise it remains active preventing the oscillations

Answer 7

1. GPCR – Receptor protein has 7 T.M helices  Change’s conformation when binding ligand 2. Conformational change allows it to associate with inactive G protein, triggering subsequent exchange of GDP to GTP, creating an active complex 3. The active complex then dissociates from the receptor to form an alpha and Beta subunit both of which are membrane anchored 4. Either the alpha subunit or the Beta/gamma dimer can activate effector proteins

Answer 8

There are ~800 human GPCRs, which makes them the largest family of cell surface receptors - 1/3 of all drugs target GPCRs The signals that activate GPCRs are very varied: 1, Photons (vision) 2. Exogenous small molecules (smell, taste) 3. Neurotransmitters (e.g. serotonin) 4. Small molecule hormones (e.g. adrenaline) 5. Peptide hormones (e.g. glucagon) 6. Proteins (e.g. chemokines)

Answer 9

Most GPCRs have a certain level of basal/constitutive activity – absence of ligand there is still a couple % activity Full agonist (native ligand) will activate the receptor to 100% Partial agonist – less than 100% Neutral antagonist suppresses activity to the basal level Inverse agonist – reduce activity below basal level

Answer 10

Popular example of GPCR - **The β2-adrenergic receptor** Class A GPCR/rhodopsin-like - First GPCR to be cloned (Lefkowitz laboratory, 1986). - Target of beta blockers (used to treat angina pectoris since 1964) - antagonist that resemble adrenaline and inhibit activation of the adrenergic receptor Image - 2 structures superimposed – Blue - Inactive / Green - active

Answer 11

β2-adrenergic receptor in complex with heterotrimeric G proteins Represents the entire Heterotrimeric G protein receptor complex

Answer 12

Variety of different classes/families of GPCRs 1. Class A – Rhodopsin like receptors 2. Class B – Larger Extracellular domain – receptor for peptide hormones (glucagon) 3. Class C – Different ligand binding domain which is coupled to a normal GPCR --\> form dimers – Metabotropic glutamate receptor 4. Class F – Even larger of Extracellular domain Note - The coupling mechanism with the heterotrimeric G protein is the same for Class A and B

Answer 13

Due to advances in **Cryo-EM** we were able to obtain structures for a variety of Class A and B GPCRs as well as GPCR + arrestin

Answer 14

Activation of heterotrimeric G protein by ligand bound GPCR (1) Agonist binding leads to a conformational change in the GPCR, which stimulates G protein coupling and GDP/GTP exchange in the G protein (alpha-subunit) (2) Active, GTP-bound, G protein dissociates into α subunit and βγ dimer (both are membrane-bound due to lipid modifications). (3) The α subunit and βγ dimer regulate effector proteins (e.g. AC, adenylyl cyclase – ATP to cAMP). (4) GTPase activity of G protein will lead to GTP hydrolysis deactivates the α subunit, which eventually reassembles back with a βγ dimer.

Answer 15

Adenylyl cyclase is an effector protein - regulated by Gα protein effector Converts ATP in cyclic nucleotide (cAMP) – removes diphosphates Adenylyl cyclase – membrane spanning protein with two intra-cellular globular domains C1a and C2a (homologous to each other) which possess enzymatic activity 1997 the globular domains coupled with alpha G protein domain were crystallized

Answer 16

2019 – full length structure was determined using Cryo-EM 12 TM helices form bundle in membrane C1A and C2A associate with each other

Answer 17

Example - Somatostatin signalling --\> activates a slow cell signalling response via PKA Somatostatin is an inhibitory hormone. - In the digestive system, it decreases gastric emptying and the release of pancreatic hormones. - In the nervous system, it decreases the release of pituitary hormones (growth hormone, thyroid- stimulating hormone, prolactin)

Answer 18

Mechanism: 1. cAMP created and binds to inactive PKA to release activated PKA 2. Active PKA enters the nucleus and phosphorylates the TF CREB 3. Activated CREB can bind to the CRE promoter element – leading to gene transcription 4. Products of gene transcription create desired effect CRE = cAMP response element CREB = CRE-binding protein (a transcription factor)

Answer 19

Example of a fast response mediated by PKA: fight-or-flight response **Fight or flight** 1. Adrenaline binds to receptor - B-adrenrgic receptor --\> activation of Heterotrimeric G-protein 2. Leads to an increase in cAMP via adenylyl kinase 3. Leads to activated PKA 4. Leads to phosphorylation of the inhibitory subunit that normally binds & inhibits the calcium channel --\> when phosphorylated it is released 5. Calcium channel open --\> allows for the subsequent influx of calcium ions

Answer 20

Phospholipase C-β is another important Gα protein effector Phospholipase C-β is a enzyme that is associated with the inner leaflet of the P.M. via its CT domain Upon activation of GPCR --\> the G-alpha subunit bound to GTP allosterically activates PLC-β which cleaves the phospholipid PIP-2 (located on the inner leaflet of the membrane) --\> releasing a soluble hydrophilic head group (IP3) and leaving behind DAG (diacylglycerol)

Answer 21

Schematic showing/elaborating on the PLC-β action 1. Diacylglycerol - remains in the membrane and associates with PKC (protein kinase C) 2. After cleavage, IP₃ acts as a secondary messenger and diffuses to the ER and associates and opens Ca²⁺ channels resulting in the movement of Ca²⁺ from the lumen [high] to the cytosol [lower]

Answer 22

Two types of receptors in the ER which are called 1. IP_s receptors 2. Ryanodine Receptors We get positive and negative feedback loops which result in waves of calcium being released 1. Positive feedback loop - IP₃ binding to IPs receptors leads to movement of Ca²⁺ into the cytosol which induces/activates other channels (Ryanodine Receptors) to activate/open – amplification of signal at lower Ca²⁺ concentrations 2. Negative feedback loop - once high calcium concentrations are reached they inhibit the activity of the channels preventing the movement of Ca²⁺ into the cytosol - Ca2+ pumps in the ER restore low Ca2+ cytosol concentration until IP3 binds again to induce a release of another wave

Answer 23

**Vasopressin-induced [Ca²⁺] oscillations in liver cells** Illustration of the wave phenomenon produced IP₃ and ryanodine receptors in ER Graph illustrates relationship between **Vasopressin concentration applied to cells and the concentration of Ca²⁺** - High Vasopressin concentration, result in increased IP3 release resulting in quicker oscillations of calcium within the cells (waves move closer together) - In response to stress, vasopressin induces liver cells to release glucose from glycogen. - The liver vasopressin receptor is a class A GPCR, and downstream signalling occurs via the PLC-β-IP3 pathway.

Answer 24

Calmodulin relays the increase in [Ca²⁺] to other proteins Concentration of calcium is sensed by calmodulin Presence of Ca2+ --\> Calmodulin binds to 4 Ca²⁺ via positive cooperativity --\> induces a conformational change whereby the linker forms a stable helix which can interact with effector molecules (e.g. CaM kinase peptide)

Answer 25

Ca²⁺/calmodulin-dependent kinases (CaMKs) CaMK II is a dodecamer, consisting of two rings of six subunits each. The subunits consist of a kinase domain and a non-catalytic hub domain, which are connected by a linker containing the CaM-binding site and an autophosphorylation site.

Answer 26

CaMKs is a large oligomeric enzyme consisting of 2 rings of 6 subunits – Hub domain, kinase domain and linker 1. Inactive kinase domain can undergo conformational change (pops out) into the tighlty associated active state - known as molecular flickering 2. Calmodulin-Ca²⁺ comes in and binds to the linker region --\> permanently moving the kinase domain to the disassociated active state – Stabilizing the active conformation 3. In the active state the kinase domain can phosphorylate targets and autophosphorylate itself --\> by autophosphorylating it is stabilizing the disassociated active state 4. Ca²⁺ may decrease and the calmodulin will dissociate from the linker but since the linker was auto phosphorylated it remains active until the phosphates are removed by a phosphatase --\> example of a form of **molecular memory** as the kinase remains active even in when Ca2+ drops

Answer 27

1. **When calcium spikes are low** (e.g. when hormone concentration is low) – there is sufficient time between the Ca²⁺ waves for the phosphorylation on the linker to be removed by a phosphatase - reduced CaMK II activity 2. **When calcium spokes are high/frequent** (e.g. when hormone concentration is high) – there is not sufficient time for the phosphatase to act to inactive the CaMK II, so we get a rapid increase in its activity (memory from the previous wave) until the system saturates

Answer 28

1. GPCR activated - activates G-alpha protein 2. Activates PC-β which results in PIP cleavage 3. DAG remains in the membrane and activates protein kinase-C 4. IP3 is released in the cytosol which interacts with Ca2+ channels in the ER, causing a wave of Calcium release 5. Ca²⁺can then bind and activate Calmodulin which in its active state can interact with various effector molecules (CaMKs)

Answer 29

GPCR variants have also been associated with particular disease states Example of a monogenic disease - congenital stationary night blindness Linked with to a constitutively activating mutation which increases GPCR activity

Answer 30

**Orthosteric site** - Binding pocket accommodating endogenous receptor ligand **Allosteric site** - binding site that is distinct from the orthosteric site Allosteric modulators are classified as negative allosteric modulators or positive allosteric modulators (NAMs and PAMs, respectively) --\> They modulate the function of the ligand in a positive or negative manner – can be endogenous (Sodium – NAM / Cholesterol – PAM or NAM) or synthetic

Answer 31

Bitopic ligand - Molecules that interact with both the orthosteric and allosteric sites, may be either agonists or antagonists. It is clear that there are multiple intermediate conformational states that exist for GPCRs (alone and in complex with their effectors) - as shown by the ternary model many different activation states and protein/ligand bound are possible – some of which have been corroborated using structural data

Answer 32

Brain structure called the **olfactory bulb** which sits above our nasal cavity There are gaps in the ethmoid bone that allow for the passage of Olfactory sensory neurons into the olfactory epithelium Specialized neurons - **Olfactory sensory neurons** (OSN) connect the olfactory bulb to the nasal cavity – goes through openings in the ethmoid bone --\> These neurons have cilia that responsible for detection odorant molecules Olfactory epithelium - most composed of OSN and support cells called sustentacular cells. This layer also contain olfactory stem cells which regenerate OSN neurons which are one of the few neurons that can be regenerated in the human body

Answer 33

Olfactory system Each olfactory sensory neuron expresses only one type of odorant receptor (OR) – each receptor is encoded by different gene product ORs constitute the largest GPCR family in mammals **~700 human** **OR genes** (~350 are functional) – the other 350 are pseudogenes which are most likely being lost during evolution **~1,200 mouse OR genes** (~800 are functional) – number of receptor genes is larger as they rely on smell much more Mice express 5-10 million OSNs – all the neurons that expresses the same kind of receptor connect to same glomeruli (region on the olfactory bulb) – 1000-fold convergence onto this brain region

Answer 34

We don’t recognize 350 different smells, instead we recognize much more than that This is possible due to the different combinations of receptors activated by a particular odorant --\> each combination generating a unique odour Odorants can interact with multiple receptors

Answer 35

1. Odorant molecule binds to OR 2. Activates heterotrimeric G-protein - in particular Gαolf activates adenylyl cyclase. 3. Adenylyl cyclase increase cAMP concentration 4. The increase in [cAMP] opens cAMP-gated Na+/Ca2+ channels – directly opens channel – leading to the influx of ions, depolarization and an action potential 5. The initial depolarization is amplified by Cl^- efflux through Ca²⁺-dependent Cl^-channels – calcium enters and binds to the Ca²⁺-dependent Cl^-channels stimulating Cl^-efflux. Grey zone – less relevant to cell signalling - explaining how only a single OR is expressed in a neuron

Answer 36

One receptor, one neuron --\> refers to the fact that OSN only express a single type of neuron How this rule is enforced is not clear but... One study in zebrafish, OR gene silencing was dependent on OR activity: signalling through G protein βγ subunits was both necessary and sufficient to suppress the expression of other OR genes - βγ subunits responsible for suppression of OR genes? (Ferreira et al. (2014), Neuron 81:847-859)

Answer 37

Photoreception – anatomy of the retina - back of the eye 1. Sclera – connective tissue 2. Choroid – vasculature – nutrients and oxygen 3. Retinal pigment epithelium 4. Photoreceptors - Cone and Rod Cells The human eye contains ~120 million rod cells - more sensitive, localized to the periphery of the retina, little color distinction. ~6 million cone cells - less sensitive, localized to the center of the retina, three different types: red, green, blue. 5. Two further layers of cells - Bipolar cells – connect light sensitive cells to ganglion cells (cells connect to the brain) Note - Strange setup of cells - Two layers of cells (Ganglion and Bipolar cells) between light source and photoreceptors

Answer 38

Rod (most sensitive) photosensor cells All photoreceptors hyperpolarise in response to light -interior becomes more negative **Dark** – GPCR called rhodopsin is inactive - Cation channels are open in the outer segment – Cell depolarization (more positive) and high release of neurotransmitter to Bipolar cells. **Light** – GPCR rhodopsin is activated, closing the cation channels - resulting in hyperpolarization of the cell --\> the net result is that no neurotransmitters are released which is interpreted by the Bipolar cell, ganglion cell and brain as light being perceived

Answer 39

There are two types of bipolar cells for cone cells: ON bipolar cells depolarize in response to light – pass on a signal OFF bipolar cells hyperpolarize in response to light – passes on a different signal A similar ON/OFF system exists for rod cells, but it involves a different type of neuron (amacrine cells). **The ON/OFF pathways are important for the detection of light increments and decrements.**

Answer 40

Signal Amplification in Rod Cells - This signalling pathway is characterized by a significant amount of signal amplification - As we can see from the cascade 1 photon absorbed by rhodopsin leads to a change in membrane potential of 1 mV (10⁶-10⁷ Na+ ions prevented from entering in one second) - Human retina has a sensitivity of around 5-10 photons

Answer 41

Return to resting state and adaptation – Move from Light to dark Four different mechanisms... 1. GAP binds to the transducin Gα subunit and stimulates GTP hydrolysis – leads to a decrease conversion of cGMP to GMP – increase cGMP 2. Closing of cGMP-gated channels leads to a decrease of cytosolic [Ca²⁺], which in turn stimulates the guanylyl cyclase to make more cGMP. 3. The falling Ca²⁺ levels activate rhodopsin kinase (GRK1) by causing the dissociation of the inhibitory EF-hand protein recoverin. GRK1 phosphorylates the cytosolic tail of rhodopsin, partially inhibiting transducin (G-protein) activation. 4. Arrestin-1 binds to phosphorylated rhodopsin, further inhibiting transducin activation.

Answer 42

GAPs are tethered to the cGMP phoshodiesterase - help to convert active G-alpha (GTP) to inactive form (GDP bound), preventing it from stimulating cGMP phoshodiesterase Transducin GTPase is the rate-limiting reaction in rod photoresponse deactivation (completed in \<0.5 seconds) - GTPase converts active G-Alpha to inactive form by change it from the GTP bound state to the GDP state --\> the presence of the GAP increase the rate of the rate-limiting reaction 1. RGS9-Gbeta5 --\> GTPase complex 2. Membrane anchor - R9AP (TM-protein) anchors complex to photoreceptor disc membranes

Answer 43

Return to resting state and adaptation – GRK1 and arrestin (Slower response) 1. GRK1 --\> Falling Ca2+ levels activate rhodopsin kinase (GRK1) by causing the **dissociation of the inhibitory EF-hand protein recoverin**. Active GRK1 phosphorylates the cytosolic tail of rhodopsin, **partially inhibiting** transducin (G-protein) activation 2. Arresting binds to the phosphorylated tail – inhibiting its activity – completely inhibits any activation of transducin

Answer 44

The entire cycle takes several minutes to complete (recovery time after photo-bleaching). Retinoid cycle doesn’t take place in the photoreceptor cell but instead in the **retinal pigment epithelium** – adjacent to photoreceptor cells

Answer 45

Arrestin is the terminating step for rhodopsin but... β-arrestins appear to be involved in signalling - desensitization, internalization, MAPK signalling and transcriptional activation This means that when the Rhodopsin in bound to β-arrestin it can itself become involved in signal transduction - different pathway

Answer 46

G-protein bias vs. Arrestin Bias Bias of the system - Refers to the fact that ligands can bind to either the G-protein bound state or the arrestin bound state depending on its bias - Bias depends on the nature of interaction with the receptor

Answer 47

1. **Biased ligand** – ligand induces a **unique receptor conformation** that results in differential coupling to the signal transduction cascade and a biased response relative to a reference agonist - different agonists or even the same agonist with different PTMs binding to different sites - e.g. Differential glycosylation of FSH 2. **Biased receptor** - modifications of the receptor influence its ability to bind ligands or transducers - ultimately influencing the response E.g. Number of phosphorylation sites on the rhodopsin tail – the receptor will be biased towards or against arrestin vs G-protein binding E.g. mutation of glutamic acid, serine or threonine residues in the c-terminus of neuropeptide Y4 receptor disrupts agonist-induced recruitment of β-arrestin 2 and receptor endocytosis 3. **Biased system** – **differential expression of transducer elements proximal to the receptor**, such as the receptor itself, β-arrestins or GRKs, as well as expression of amplification cascades distal to the receptors - e. g. cell increases GRK expression leading to more phosphorylation and β-arrestins binding - β-arrestin bias E.g. Certain agonists targeting the **dopamine 2 receptor** (D2R) have **different effects in the striatum and prefrontal cortex**, which could be related to the differential expression of β-arrestins and GRKs across brain regions **Note - GPCR is very conformationally labile** – very mobile molecule. Hence, depending on what molecules bind we get different conformations

Answer 48

Class of Enzyme-coupled receptors - Ligand binding to the receptor activates an intrinsic catalytic activity of the receptor 1. Receptor tyrosine kinases 2. Tyrosine kinase-associated receptors - similar but the tyrosine kinase is a separate polypeptide (different gene) that is permanently associated with the receptor 3. Receptor serine/threonine kinases 4. Receptor-type tyrosine phosphatases (not discussed) 5. Receptor guanylyl cyclases (not discussed)

Answer 49

Unlike in GPCRs (associates with heterotrimeric G protein), the catalytic activity of enzyme-coupled receptors (e.g. tyrosine kinase) is associated permanently with the receptor, either as part of the same polypeptide chain or non-covalently.

Answer 50

Receptor tyrosine kinases (RTKs) - Receptor tyrosine kinases are single-span transmembrane proteins (single alpha-helix traversing the membrane) with an extracellular ligand binding domain and an intracellular tyrosine kinase domain. - Ligand binding results dimerization allowing for trans-autophosphorylation of the receptor --\> generating binding sites for signalling proteins

Answer 51

The human genome encodes 58 RTKs - **Extracellular** ligand binding domains is varied - But the **intracellular** is always a intracellular tyrosine kinase

Answer 52

Examples of signalling molecules that signal through RTKs. 1. Epidermal growth factor (EGF) - important 2. Insulin 3. Nerve growth factor 4. Fibroblast growth factor

Answer 53

RTKs are frequently over-expressed or mutated in cancer

Answer 54

Receptor activation is commonly studied by **Western blotting using antibodies against phosphotyrosine** (anti-pTyr). Procedure --\> Cell treated with ligand, cell lysed, immunoprecipitated, run on SDS-page and blotted with antibody against phosphotyrosine. Increasing ligand conc. results in increased tyrosine phosphorylation **But!** To ensure that the total level of RTKs is not changing we... Control for the total level of receptor – you strip the blot and **re-probed with an antibody for the receptor itself.** Hence, we can safely deduce that the increase in phosphotyrosine is due to receptor trans-autophosphorylation rather than an increase in receptors

Answer 55

Yes, many RTKs are dimerised by their ligands. The proximity of the kinase domains in the RTK dimer favours trans-autophosphorylation.

Answer 56

Epidermal growth factor (EGF) is a monomer. When it binds to monomeric EGF receptor, a dimerization arm is in receptors is exposed, which promotes EGFR dimerization. Binding of EGF to monomeric EGF induces a conformational change which exposes the dimerization arm which allows EGFR to dimerize

Answer 57

Insulin receptor is a covalent, disulphide-linked dimer --\> receptor is already dimerized! Insulin receptor is composed of the polypeptide chains (alpha and beta). The dimers are held together by disulphide bridges **Activity** When insulin binds, the extracellular domain changes conformation which ultimately brings the two intracellular kinase domains together allow for phosphorylation **Takeway** - Highly variable 1. Some RTKs are non-covalently associated in the absence of ligand (e.g. DDRs) 2. Some RTKs require higher-order clustering for full activation (e.g. DDRs) 3. Some RTKs require co-receptors (e.g. FGFRs)

Answer 58

Heparan sulphate is an essential co-receptor for fibroblast growth factor

Answer 59

Kinase Domain – Active v.s. Inactive – example Insulin Receptor Kinase **Inactive state** – Activation loop (green) blocks the activation site – Tyr1162 adopts a pseudosubstrate conformation, meaning it occupies the site where the actual substrate tyrosine would be located **Active state** - the activation loop undergoes a conformational change so that it no longer blocks the active site + activation loops three tyrosine residues are phosphorylated preventing returning to the inactive state - Substrate tyrosine (other kinase activation loop) occupies the active site

Answer 60

The unphosphorylated activation loop is predominantly in the inactive conformation – blocking the active site. **But there are transient excursions out of the active state** When dimerization occurs… For transphosphorylation to occur, two kinase domains with the activation loop in the active conformation have to meet; this is much more likely if the kinase domains are in close proximity --\> One loop acts as the substrate the other free's the active site Two requirements are needed for transphosphorylation 1. **Transient excursion** of the loop so that it can be phosphorylated – but once one is phosphorylated it is locked (untill phosphatase removes it) so it can easily phosphorylate the other 2. **Two Kinase are in close proximity** to each other – dimerization Note - Auto-phosphorylation of the activation loop is the first step in the activation of most RTKs --\> this followed by phosphorylation of other sites in the cytoplasmic domain

Answer 61

Best studied receptor EGF - does not follow the classical mechanism just described 1. Dimerization of the inactive monomers in presence of EGF 2. When dimerization occurs, they form a asymmetric dimer - one kinase (activator) allosterically activates the receiver 3, Activation results in the autophosphorylation of the C-terminal tail not in the activation loop

Answer 62

Once the Receptor is phosphorylated, the phosphorylation sites become docking sites for downstream mediators and effectors

Answer 63

SH2 (and PTB) domains recognize phosphotyrosines The human genome encodes ~100 SH2 (Src homology region 2) domains, which mediate pTyr binding. **Structure** Small globular domain with a **conserved pocket** that recognizes and bind phosphotyrosine as well as a **specificity pocket** which accommodates an apolar residue (located 3 residues upstream)

Answer 64

Modular architecture --\> refers to the fact the signalling protein are composed of different domains (i.e. lego blocks), each providing it's own specificity E.g. SH2 domains are found together with other domains, for example PH and C2 which allow these mediators/signalling molecules to recognize the plasma membrane Why is it useful? The modular architecture enhances specificity (e.g. membrane targetting/binding domains) and allows the formation of multiprotein signalling complexes – target multiple proteins at the same time

Answer 65

Mammals have several Src family kinases - Rous sarcoma virus has captured and integrated the tyrosine kinase into its genome but a truncated version - viral oncogene (promotes tumour formation) **Inactive form -** Inactive form is autoinhibited allosterically by a SH2 domain which recognizes a phosphotyrosine at the C-terminal - this binding forcrs Src tyrosine kinase in a conformation where the C-helix is in its Up position + the activation loop is blocking the active site, rendering the active site inactive **Active form** - Phosphatase removes the phosphate of the C-terminal Tyr 527, preventing SH2 binding and thus resulting in a conformation in the active site (C-helix down + active loop swings out) --\> rendering the receptor active Note - **V-src** lacks the regulatory C-terminal region so the SH2 domain does not recognize phosphotyrosine and thus the Src Kinase remains in the active state

Answer 66

Yes, a whole host of signalling pathways are activated but there will be a focus on three in the lecture course

Answer 67

Ras is a small GTPase – Central to Ras-MPK pathway Structural difference - GTP and GDP bound states The **switch helix** is one of the regions that changes most between the GDP- and GTP-bound states. In the GTP-bound state of Ras, the switch helix is able to interact with Ras effectors

Answer 68

**Ras – Molecular Switch** Two forms 1. **Off state** – GDP bound - created by intrinsic GTPase activity of the GTPase but process is accelerated by GAPs 2. **On-state** – GTP bound - created by an exchange of nucleotides – GDP leaves and GTP binds which is catalyzed by GEF

Answer 69

Ras is a member of a superfamily of small GTPases Three members of the Ras family which is part of a larger family of GTPases 1. H/K/N-Ras 2. Rheb 3. Rap1 Rho family GTPase - change the cytoskeleton in response to the cell surface

Answer 70

Digression: Rho family GTPases regulate actin dynamics

Answer 71

Signal amplification by kinase cascades Benefit - allows for significant amplification as each kinase can activate/act on multiple kinases

Answer 72

Yes, There are a multitude of MAPKKKs, MAPKKs and MAPKs In order to create some order scaffold proteins are used.

Answer 73

Scaffold help organise the different MAP kinase pathways ensuring that correct output is produced by organising all the relevant players

Answer 74

c-fos is involved in the sustained input detector system Sustained mitogen signals through Ras-MAP kinase pathway activating ERK leads.... 1. Slow response - Fos1 transcription via TCF and SRE 2. Fast response - Phosphorylation of Fos1 via an activated ERK Only if Fos1 is stabilized by phosphorylated and is able associate with c-Jun to form the AP-1 TF, do we get cell division Note - the signalling pathway is constructed in this way so that a brief mitogen input doesn’t lead to cell division but sustained mitogen signalling does drive cell division as the Fos1 can get transcribed and phosphorylated.

Answer 75

This pathway is controlled by… **Multiple negative feedback loops** Two examples of -ive Feedback loops 1. **Fast response** - Erk phosphorylates Sos breaking Grb2-Sos interaction 2. **Slow response** - One of the gene’ transcribed as a result of Erk action is MKP (phosphatase) which directly dephosphorylates Erk MKP = MAPK phosphatase - (family of dual-specificity phosphatases, i.e. act on pSer/Thr and pTyr)

Answer 76

1. Ras activation by RTKs is short-lived (intrinsic activity + GAPs) + RTKs are inactivated by specific phosphatases 2. The MAP (mitogen-activated protein) kinase pathway amplifies the signal and conveys it from the cell surface to the nucleus. 3. Only a sustained input drives cell division 4. Various positive and negative feedback loops exist to modulate the strength and duration of the signal. 5. Scaffold proteins help prevent crosstalk between parallel MAP kinase modules. 6. Due to the importance of Ras-MAP in cell division - Ras and B-RAF are proto-oncogenes. Ras mutations are found in ~15% of human cancers. B-RAF mutations are found in ~60% of melanomas

Answer 77

Signalling by cytokines (JAK-STAT pathway) Cytokines are recognized by receptors that are similar to RTKs – Tyrosine Kinase associated receptors (Tyrosine kinase is a separate protein bonded to the receptor – e.g. JAK)

Answer 78

Mechanism 1. Ligand binds – dimerization 2. JAKs phosphorylate each other as well as receptor 3. Creates docking sites for mediators (STAT) 4. STAT become phosphprylated and dissociates 5. After dissociation, STATs form a dimer which enters the nucleus in order to influence transcription Note - Phosphorylation is required for dimerization and thus activity

Answer 79

TGFβ – important for development and wound healing Mechanism 1. TGFβ dimer binds to receptor - forming a heterodimer between type 1 and 2 TGFβ receptors 2. Type 2 receptor phosphorylates type 1 receptor (serine/threonine kinase) 3. Active Type 1 kinase phosphorylates a Smad2/3 (TF) which can form a dimer with Smad4 4. Moves into the nucleus where it binds to TGFβ cis-regulatory sequence act as a transcription regulatory complex

Answer 80

- **RTK pathways** are longer & involves more players - **STAT and SMADs** pathways are much more direct and quicker – involve 1 TF which is phosphorylated by the receptor which can then translocate to the nucleus in order to influence transcription --\> faster response

Answer 81

The two mediators present in EGF signalling – **PI3K and PLC-γ**

Answer 82

PIP2 cleavage by PLC-γ generates the second messenger IP₃ Pathway 1. Activated EGFR/RTK is bound by PLC-γ which has a 2xSH2 domain 2. Activated PLC-γ causes the cleavage of PIP2 - releasing IP3 and diacylglycerol 3. IP3 interacts with Ca²⁺channels in the ER, releasing Ca²⁺, which is sensed by Calmodulin and thus Calmodulin dependent kinase 4. Diacylglycerol interacts with protein kinase C activating it (binds Ca²⁺ as well) - Identical to what has been described for GPCR

Answer 83

Phosphoinositide 3-kinase produces PIP₃ PIP2 is composed of 2 fatty acids, glycerol and Inositol sugar (each hydroxyl group in the sugar ring can be phosphorylated) – PIP2 – biphosphate (4,5) To generate PIP3, PIP2 is phosphorylated by PI 3-kinase at position 3.

Answer 84

PIP₃is recognised by pleckstrin homology (PH) domains

Answer 85

1. Activated RTK bound by survival signal 2. Activated PI-3 kinase binds to activated RTK (allosteric activation) leading to conversion of PIP2 to PIP3 3. PIP3 acts as a docking site for 2 different kinases (PDK1 and Akt) 4. Akt become phosphorylated by PDK1 and mTor 5. Active Akt dissociates and phosphorylates a protein called bad 6. Inhibitory action of Bad terminates releasing an active apoptosis inhibitory molecule (Bcl-2/Bcl-xl) --\> P-Bad binds to 14-3-3 protein 7. Bcl-2/Bcl-xl inhibit apoptosis - If signal is not present the cell would undergo apoptosis

Answer 86

1. **Dephosphorylation** - Carried out by protein tyrosine phosphatases (PTPs) 2. **Endocytosis** – remove the receptor from the cell surface --\> Receptor recycling or degradation Note - Does not always terminate the signal as signalling can continue in endosomal compartments  same topology

Answer 87

Ubiquitination of activated EGF receptor by the E3 ligase Cbl When EGF becomes activated and phosphorylated it can recruit both Grb2 and Cbl Cbl has a E3 ligase that can ubiquitinate the receptor signalling for internalization – terminating the signalling

Answer 88

Yes there is Overlap of GPCR and RTK signalling pathways ("cross-talk") Example.... 1. Phospholipase C/IP3/diacylglycerol pathway are stimulated by both receptors 2. Arrestin can itself be a signalling mediator and can turn on MAP kinase pathway 3. Recent publication – activated RTK can influence G-proteins as well

Answer 89

Different feedback loops determine signalling outcomes Famous experiment - PC12 cells expressing both EGF and NGF were exposed to either EGF or NGF resulted in different outcomes a) EGF – resulted in proliferation b) NGF – resulted in differentiation EGF and NGF both activate the Raf-MEK-Erk pathway in PC12 cells but different feedback loops at play **EGF** - A **negative feedback loop** downstream of EGFR produces **transient activation** of the MAPK pathway, resulting in cell proliferation - Erk phosphorylates SOS which inhibits Raf via Ras **NGF** - A **positive feedback loop** downstream of TrkA (NGF receptor) creates a bistable system, resulting in cell differentiation and neurite outgrowth --\> Erk phosphorylates protein kinase C which phosphorylates Raf stabilizing the system meaning that the **signalling remains activated**

Answer 90

Erk is responsible for both c-Fos transcription as well as phosphorylation Hence, in EGF as we only get a brief stimulation of ERK only small quantities of c-Fos will be stimulated resulting in proliferation. Whereas for NGF the sustained signalling results in increasing quantities of stable pc-Fos created stimulating differentiation **Basically different quantities of pc-Fos produced as shown by graphs in figure**

Answer 91

Possible ideas 1. Another player involved – e.g. another player that activates with NGFR which allows PKC activity – effect on PKC that is unique to NGFR 2. Possibly internalization?

Answer 92

Different feedback loops determine signalling outcomes Introducing players to modulate feedback loops in order to examine the outcome 1. Add PMA to cells – stimulates PKC  even EGF will lead to differentiation as we have artificially stimulated PKC  resulting in a NGF like profile 2. Add Gö7874 inhibits PKC action - converts the normal NGF profile (differentiation) into the EGF profile of proliferation as it inhibits the +ive feedback loop that sustains the signal

Answer 93

'YESSSSS' - Borat accent

Answer 94

Hohenester’s own research of a specific RTK – DDR1/DDR2 DDR1 and DDR2 are 50% homologous and are thought to function in a similar way

Answer 95

Authors were searching for proteins related to the insulin receptor and screened a human placental cDNA library with a 32P-labelled antisense oligonucleotide against a region that is conserved in many tyrosine kinases Basically, probing a cDNA library for new kinases They discovered a new RTK with a discoidin-like domain in the extracellular region --\> at the time it was known as an orphan receptor - unknown ligand

Answer 96

Collagen --\> Surprise that this structural protein also had a functional role as a ligand

Answer 97

Activation kinetics was very unusual **Experiment** Cells expressing the receptor are exposed to the activating ligand for different time periods and the lysed cell is probed for antibodies against phosphotyrosine (activation) and followed by probing for receptor using antibodies (control) **Results** It takes 2-4 hours of ligand exposure to obtain saturation of autophosphorylation of the discoidin domain receptor Plus the levels don’t of phosphotyrosine don’t drop quickly (sustained for several days)

Answer 98

Physiological and pathological roles of DDRs Discovered from gene knock-out studies and studies in human patients **DDRs are involved in Tissue and organ development** 1. Mammary gland (DDR1) 2. Inner ear, kidney (DDR1) 3. Long bones (DDR2) **Range of Pathologies where DDRs are associated** 1. Atherosclerosis (DDR1) 2. Kidney disease (DDR1) 3. Arthritis (DDR2) 4. Fibrosis (DDR1 and DDR2) 5. Cancer (DDR1 and DDR2)

Answer 99

How does collagen bind to the DDR receptor? How does collagen activate the receptor? How is it regulated?

Answer 100

First thing required before performing further experiments is a ligand binding assay 1. Collagen (ligand) is immobilized to wells in the 96 well plate and the receptor is added with increasing amounts 2. Add an antibody against the receptor --\> so if receptor binds the antibody will also bind – antibody is coupled to an enzyme that creates a visual color when substrate is added (coupling) --\> colour change is an indicator of receptor binding 3. Plot O.D. (color) against receptor construct added (His-DDR2) - we get an asymptotic curve Results Collagen I – high affinity but when heated/denatured affinity is lost. Furthermore, other extracellular proteins (Laminin and fibronection) are also tested - low affinity

Answer 101

Collagen is a highly repetitive structure – glycine at every 3rd position to allow for the superhelix to be created + other common A.A. is proline Not easy to recombinantly form collagen + break into small peptides. Thus, making it hard to run experiments to determine the binding site **BUT!!!** **Collagen toolkit** allows us to create small collagen fragments that retain triple helical structure Native guest sequence (actual sequence) is flanked by host sequences (synthetic sequences Gly + Pro – high propensity to form triple helix) Able to create a library of different collagen fragments that harbour a region of the native sequence

Answer 102

Identification of the DDR2 binding site in collagen Collagen toolkit library containing 56 peptides that span the entire native collagen sequence Used the ligand binding assay to scan the entire library Results? Recognition sequences overlapped between two peptides, hence producing the two central peaks -- sequence given in attached image (key residues highlight - O = hydroxyproline)

Answer 103

Having identified the key collagen sequence – the peptide was co-crystallized with DDR2 DS domain (extracellular region) M, F and O play an important role - substitution of these residues eliminate binding Important residues come from 2 different chains in collagen. Hence, explaining why the denaturation of the triple helical structure results in a loss of binding - residues need to be present and positioned in the specific conformation relative to eachother

Answer 104

Bone disorder related to DDR2 – SMED Defect in the growth of long bones and is accompanied by abnormal calcification

Answer 105

Examining cells expressing DDR2 – either WT or mutants Cells were stained for DDR2 and Ras - Ras is used simply as a marker for the Plasma membrane Merge – combination of DDR2 and Ras stain  allows for identification of any differences Results 1. WT and E113K are **normally trafficked** 2. Kinase domain mutants of DDR2 are **not trafficked to the surface** - these mutants remain interior (likely in ER) explaining why they are disease causing

Answer 106

The E113K mutation abolishes collagen binding E113 --\> Glutamic acid is one of the key residues involved in hydroxyproline binding

Answer 107

One way to study RTK dimerization can be studied is using disulphide cross-linking Idea – make number of mutants where you replace residues with Cys in the linker and transmembrane region Subsequently, you expose mutants to the ligand, extract them and run them on non-reducing SDS-page gel to detect which species dimerize (only be present as dimer if disulphide bridge formed) allows us to identify mutants that readily cross-link Example using EGF

Answer 108

Performed the cross-linking experiment in the presence and absence of ligand **Results** 1. Strong dimerization signal for all the mutants created in presence and absence of collagen Meaning that even in the absence of a ligand DDR is a dimer + there is efficient cross-linking --\> different to EGF mutants on previous card 2. Cross-linking had no effect on DDR1 activation – all were able to bind to phosphotyrosine antibody - normal behaviour

Answer 109

Collagen induces the formation of large DDR1 signalling clusters Absence of Ligand (unstimulated) - DDR1 forms clusters Presence of ligand (10 min) - DDR1 redistribute to form peculiar worm like tubular structures – unusual for RTKs Clustering is not yet fully understood

Answer 110

DDRs have a very long juxtamembrane region – unstructured BUT! Only the last quarter of the JM region is required for DDR1 activation Experiment - Deleted different regions of the JM region and examine impact on DDR1 activation (Phosphotyrosine antibody) JM4 was identified to be required for DDR1 activation as its deletion abolishes activation JM4 must carry out some functional role

Answer 111

Crystal structure illuminated importance of JM4 - this region inserts itself into a cleft in the kinase domain above the activation loop. JM4 region **reinforces autoinhibition** by the activation loop - basically also sterically blocks the kinase in the inactive state – preventing ATP binding + positioning of C-helix for kinase activity

Answer 112

**Experiment A)** --\> Expose JM4Kinase-WT and mutants to ligand for different time periods and run on native-PAGE Results 1. JM4K-WT showed increase in movement in the first 5 minutes and after 3 hours --\> increase in movement due to phosphorylation as it increases the charge of the molecule BUT! --\> JM4K tyrosine mutants (residues in JM4 loop) --\> did not exhibit this increase in movement --\> Not getting phosphorylated **Experiment B)** --\> Run a similar experiment but stain the gel with site-specifc tyrosine antibodies --\> showing us that JM4 tyrosine residues (orange) are phosphorylated before activation loop (green) **What the fuck does this all mean???** Tyrosine residues in the JM4 region become phosphorylated quickly, followed by the slow phosphorylation of the activation loop --\> unusual as normally the activation loop become phosphorylated first in RTKs JM4 phosphorylation is required first before activation loop phosphorylation --\> removal autoinhibtory effect

Answer 113

Graph shows us that only normal Michaelis-Menten are obtained when both JM4 and A-loop are phosphorylated There is some reduced activity when JM4 is only phosphorylated This holds true for both substrates – Peptide and ATP

Answer 114

**JM4 phosphorylation is required for DDR1 activation** 1. JM4 Y569F mutation didn’t have a significant effect on receptor activation 2. JM4 Y586F - reduction in activity 3. JM4 Y569F/ Y586F - abolished activity – receptor is basically inactive

Answer 115

1. A triple-helical peptide library was used to identify the DDR binding site in collagen - normally recombinant dissection is used 2. X-ray crystallography has revealed the details of the DDR-collagen interaction 3. Disulphide cross-linking experiments ruled out ligand-induced dimerization or conformational changes as mechanisms of DDR activation 4. Using Super-resolution microscopy has revealed large-scale DDR redistribution in response to collagen binding - tubular structurs 5. Structural and biochemical experiments have shown that JM4 plays an important role in DDR autoinhibition and activation --\> act as a second level of control of DDR above simply the activation loop.

Answer 116

Decrease in odorant - GPCR is no longer activated 1. The cAMP concentration lowers as it hydrolyses to AMP 2. Alpha subunit of G-Protein effectively terminates its own activity, hydrolysing GTP to GDP, re-joins to the beta and gamma subunits and retains its resting (OFF) state 3. Cell regains its electrical neutrality and maintains Ca2+ homeostasis by pumping Ca2+ out of OSNs by sodium-calcium (Na+/Ca2+) exchangers, voltage-gated chloride channel (ClCn) and transport proteins Ca2+ ATPase

Answer 117

Adaptation / Short-term adaptation (STA) refers to a d**ecrease in the response to the odors by OSNs** - reduction in excitation even if odorant is still present CNG - cyclic nucleotide-gated channel OSN excited --\> Influx of Calcium 1. Calcium bound calmodulin (Calm) binds to CNG channel reduces channels sensitivity to cAMP, thus CNG channel closes 2. Ca2+/Calm activates enzyme Phosphodiesterase (PDE) which transforms cAMP to 5’AMP - cGMP is also produced during odorant stimulation - [cGMP] decreases CNGs close 3. cGMP-activated PDE2 hydrolyses both cAMP and cGMP, hence terminating their action 4. Regulators of G-protein Signaling (RGS) also suppress GPCR mediated signals by accelerating the hydrolysis of GTP bound to the Gα subunit

Answer 118

**Desensitization** involves loss of responsiveness by ORs in the repetitive presence of odorants or stimulus Mechanism includes **phosphorylation** of the ORs resulting in uncoupling from its heterodimeric Gα-olfprotein --\> phosphorylation also signals for their internalization to the cytosol and down-regulation of the cellular components of OR **PKA, PKC and GPCR specific kinases (GRKs),** phosphorylate the serine and threonine residues present in the intercellular loops and C-terminal regions of ORs. Note - GRKs promote binding of arrestins, cytosolic co-factor proteins, causing steric uncoupling of the receptor and G-protein

Erhand Hohenester - Cell Signalling Flashcards

(167 cards)