Cell Signalling Flashcards

Question

What happens when you combine a feedforward loop with an AND gate

Answer 1

> sustained input detector: ensures output is only generated from a long-lasting response > applies to when input generates a fast and slow branch of signal and both are required via AND gate to generate output > spike of signal: only fast branch initially present (no output); slow branch follows and fast is no longer present (no output) > sustained: both are present at a time so output will be generated

Answer 2

preventing aberrant cell division from spike in mitogen signal > in absence of signal, c-fos gene is not transcribed > c-fos protein is not stable unless P by active Erk (MAPK), any existing c-fos gene will be degraded so no c-fos protein > mitogen signal activates Erk > Erk P c-fos (fast) > Erk leads to expression of c-fos gene via TFs TCF and SRF (slow) >w/brief pulse of mitogen signal fast pathway has little to act on because no c-fos protein in the cell yet > slow pathway then leads to expression of c-fos gene, but by the time the c-fos protein is made, mitogen signal is over, so there is no active Erk and no P c-fos so protein is degraded >sustained mitogen signalling, pathways are activated for longer, you have active Erk when c-fos protein is made. c-fos is then P and stabilised and can go on to act as a TF for further genes involved in cell growth & division

Answer 3

> 7TMD >ligand binding causes conformational change of GPCR which allows inactive heterotrimeric G-protein to bind >leads to exchange of GDP for GTP on G -a domain, activating the G protein > dissociation of heterotrimeric protein to a + b, y subunit – both go on to activate other proteins in the pathway > ~800 human GPCRs: largest family of cell surface receptors > GPCRs are a major target of small-molecule drugs in clinical use

Answer 4

Photons (vision) Exogenous small molecules (smell, taste) Neurotransmitters (e.g. serotonin) Small molecule hormones (e.g. adrenaline) Peptide hormones (e.g. glucagon) Proteins

Answer 5

>due to multiple conformational states & linked equilibria because: receptors are dynamic – naturally switch between active/inactive conformation, so no receptors are truly off – always a level of basal activity even without ligands

Answer 6

>Without ligand, they just exist with less time in active conformation > with a ligand, if: - receptor on to 100% activity, L = full agonist - receptor on to less than 100% activity, L = partial agonist - binding doesn’t result in increase/ decrease of the receptor activity, L = neutral antagonist -binding results in decrease in activity, L = inverse agonist

Answer 7

ß-adrenergic receptor (Class A, rhodopsin-like) -normal agonist = adrenaline -target of beta-blockers (used to treat angina pectoris) -propanolol inhibits its activity = inverse agonist

Answer 8

1. agonist binding leads to conformational change in the GPCR allowing heterotrimeric G protein to bind 2. active GPCR stimulates GDP/GTP exchange in G-protein by GEF. GTP binding also weakens the G-protein interaction w/receptor. 3. Active G-protein dissociates into a + by dimer (both membrane-bound in inner leaflet of PM due to lipid-modifications) 4. a and by dimer can regulate downstream effector proteins (eg. adenylyl cyclase, PLC, ion channels) 5. GTP hydrolysis by GAPs deactivates the a subunit, which can reassemble w/ by dimer

Answer 9

> important G-protein effector > membrane-spanning protein > has intracellular loops, C1a and C2a that bind to G-protein and contain enzymatic activity

Answer 10

> forms cAMP from ATP catalyzing the formation of a cyclic phosphodiester bond between OH in ribose and the a-P, resulting in Ppi leaving > can be acted on by phosphodiesterase to hydrolyze the bond to make AMP

Answer 11

> diffuses in the cell to generate a response (2nd messenger; 1st one is the ligand that binds GPCR) > activates cAMP-dependent PKA -inactive state: pkA catalytic subunit is in complex with regulatory subunit, blocking the active site -cAMP binding to PKA regulatory subunit allows catalytic subunit to dissociate and perform its functions

Answer 12

> somatostatin expression > active PKA translocates to the nucleus > P CRE-binding protein (TF), allowing it to bind to cAMP response element (cis-regulatory element upstream of somatostatin gene) > leads to transcription of somatostatin (inhibitory hormone) digestive system: decreases gastric emptying and the release of pancreatic hormones nervous system: decreases the release of pituitary hormones (GH, TSH, prolactin)

Answer 13

> fight or flight response > PKA P Rad (an ion channel inhibitor), leading to influx of ions, initiating fight-or-flight response

Answer 14

> important G-protein effector >peripherally associated w/membrane via CT domain >ligand binding to GPCR activates G-protein which activates PLC >hydrolyzes PIP2 (inner-membrane glycerol phospholipid) to 1,2-diacylglycerol and IP3

Answer 15

> second messenger > binds to and activates open ryanodine receptors in the ER membrane which alow CA2+ influx from ER into the cytoplasm >Ca2+ normally regulated to be low in the cytoplasm

Answer 16

> ryanodine receptors also have calcium binding sites: -high affinity BS: activate the receptor -low affinity BS: inhibit the receptor positive: > the first wave of Ca release from the receptors binds to high affinity calcium site, activating neighbouring receptors, which amplifies the signal & causes Ca-induced-Ca-release, resulting in high Ca in the cytoplasm negative: > high concentration Ca, some can now bind to lower affinity Ca BS, inhibiting the receptor, allowing low Ca concentration restored in the cytoplasm

Answer 17

> High levels of Ca activate Calmodulin (cytoplasmic molecule w/2 lobes) > when no Ca bound = floppy dumbell shape > when Ca reaches a certain threshold = leads to positive cooperative binding of Ca (2Ca in each lobe) > Ca binding causes conformational change, allowing it to bind & activate many other downstream targets eg. ion channels, kinases > Calmodulin relays the increase in [Ca2+] to other proteins

Answer 18

> CaMK dodecamer - 2x 6 subunit rings stacked on top of each other -kinase domain + non-catalytic hub domain connected by linker w/ CaM BS and autoP site -inactive form: kinase domain is contained in the ring -in eq w/active form where kinase can pop out and regulatory region is accessible -active: CaM can now bind to CaM-BS in the regulatory region keeping CaMK active -active kinase can also autoP itself at the P site, preventing the kinase domain from repositioning back to inactive form -occurs in step-wise process until all 6 kinases are out, all regulatory region is bound to CaM and P

Answer 19

> during the time between Ca spikes, CaM can leave the regulatory region & P can drop of the phosphorylation site, returning each kinase subunit to the inactive form -low frequency Ca oscillation does not lead to activation of kinase because there is enough time between interval of spikes for each kinase to return to the inactive form -high-frequency Ca oscillations = not enough time for kinase to reset, gets more active until all 6 subunits are fully active

Answer 20

> olfactory epithelium at the back of the nasal cavity >contains olfactory sensor neurons with cilia pointing into the nasal cavity = where odorants are recognized > OSN connected to olfactory bulb via region in brain called ethmoid bone > olfactory bulb has the processing units to process signals from sensory neurons to give the perception of smell

Answer 21

> OSN expresses only one type of OR on its cilia to receive signals from odorants > signals from same types of OR converge in the brain in the olfactory bulb > More receptors = can differentiate more smells ~700 human OR genes (350 functional) ~1,200 mouse OR genes (800 functional)

Answer 22

> Odorant must be vaporised into gas phase to reach the sensors in the nose

Answer 23

different combinations of odorants recognised

Answer 24

-odorant-bound OR activates G-olf which activates adenylyl cyclase (AC) which converts ATP/cAMP -increase in cAMP opens cAMP-gated Na+/Ca+ channels (fast response) causing depolarization -depolarization is amplified by the efflux of Cl- through Ca2+-gated-Cl- channels -cAMP also activates PKA which is involved in the gene transcription process of "one receptor per neuron" in OSN (slow response)

Answer 25

2 types of photoreceptors in the retina: -rod cells: highly sensitive to light, function in night vision, peripheral (~120m) -cone cells: less sensitive, central, detect wide spectrum of light photons, responsible for colour vision (~6 million)

Answer 26

-membrane stacks at the back of the structure -highly metabolically active w/lots of redox species causing damage requiring a lot of repair -protected by the retinal pigment epithelium -synapse at the other end, connecting photo receptors to bipolar cells, which connect to axons of the optic nerve feeding information to the brain

Answer 27

> has rhodopsin in the membrane stacks: opsin (GPCR) + retinal (chromophore) > aldehyde group of retinal forms Schiff base linkage to Lys in opsin > before photon hits, 11-cis-retinal configuration > light isomerizes into more thermodynamically stable structure (all trans) >trans = more extended structure so pushes against the opsin GPCR, causing overall conformational change of Rhodopsin to Meta II form which allows transducin (Gt) to bind

Answer 28

absence: -guanylyl cyclase converts GTP to cGMP -opens cGMP-gated cation channel -ion flow-depolarization - high rate neurotransmitter release - bipolar cells pass input that it is dark presence: -retinal of rhodopsin is extended -push on opsin GPCR -rhodopsin conf changes to Meta II -activate Gt - activate cGMP phosphodiesterase - degrades cGMP to GMP -cGMP gate cation channels close -no ion flow-cell hyperpolarizes -no neurotransmitter release -bipolar cells pass input that there is light

Answer 29

> get less signal in response to light

Answer 30

1 rhodopsin molecule absorbs 1 photon V 500 Gt molecules activated V 500 cGMP phosphodiesterases activated V 10^5 cGMP molecules hydrolyzed V 250 cation channels close V 10^6-10^7 Na+ ions per second are prevented from entering the cell for 1s V membrane potential is altered by 1mV

Answer 31

GAP-induced GTP hydrolysis on transducin >GAPs bind to activated Gt tethered to cGMP PDE > GAP binding forms GTPase complex, stimulates GTP hydrolysis on Gt, deactivating it > Gt dissociates from PDE > PDE deactivation so cGMP not converted to GMP > cation channel back to open >depolarization >high rate of neurotransmitter release > no signalling to indicate presence of light

Answer 32

closing of cGMP-gated channels leads to decrease of cytosolic Ca 1. stimulate guanylyl cyclase to make more cGMP 2. activate rhodopsin kinase (GRK1) by causing the dissociation of the inhibitory EF-hand protein recoverin 3. GRK1 P the cytosolic tail of rhodopsin, partially inhibiting Gt activation (prevent G protein binding) 4. arrestin-1 binds to P rhodopsin, further inhibiting Gt activation 5. after the signal turns off, end up w/P Meta II w/bound arrestin-1 and retinal in trans form - needs to be reset to return retinal to cis form again

Answer 33

>retinal is hydrolysed from opsin -retinal is taken into the retinal pigment epithelium -throughout a series of enzymatic reactions, retinal will be converted back to cis form and return back to rod cells, where it forms Schiff base w/opsin, resetting the rhodopsin

Answer 34

> arrestin function not restricted to rhodopsin/signal inhibition > ß-arrestins = signalling mediators (can induce signals themselves) > can also bind to GPCR-like G proteins but only when GPCR is P >cause internalization of GPCR & can start pathways eg. MAPK signalling pathway or transcriptional activation

Answer 35

* occurs when there's many ligands for one receptor and can induce diff types of responses * the type of response the receptor is going to preferentially produce depends on the type of ligand-bound, the state of the receptor, or the amount of downstream molecule transcribed

Answer 36

>Unlike in GPCRs, the catalytic activity is associated permanently w/receptor, either as part of the same polypeptide chain or non-covalently. > Has ligand recognition domain on outside of PM, and a catalytic domain on the inside

Answer 37

* single-span TM proteins w/ extracellular ligand binding domain and an intracellular tyrosine kinase domain. * Exist as monomers in PM until ligand binds, resulting in dimerization * Proximity of 2 catalytic domains result in trans-autophosphorylation of the receptor, which then become docking sites for mediators that lead to downstream signals * human genome encodes 58 RTKs in ~20 diff families

Answer 38

- Ligand binding domain = different across members - Catalytic domain = the same across all members - each RTK respond to different signals but will always result in tyrosine P at the end

Answer 39

* Either due to mutation that causes RTK to be more active or over-expression of RTK in cancer cells, resulting in the presence of more RTK on cell surface * good cancer drug targets -Traztuzumab = monoclonal antibody that interferes w/Her2 (1/4 EGFR), used to treat breast and stomach cancer -Gefitinib = tyrosine kinase inhibitor specific for EGFR. used to treat cancers in which EGFRs are over-expressed or mutated

Answer 40

commonly studied by Western blotting using antibodies against pTyr 1. Cells expressing an RTK are treated w/ increasing amount of activating ligand 2. Lyse cells w/ detergent, run on gel & transfer to membrane, probe membrane w/ chemiluminescent antibody against phosphotyrosine 3. Result shows increased amount of phosphotyrosine in cells w/ more amount of activating ligand – correspond to receptors being activated 4. Must also run an experiment to ensure that there are equal amounts of receptors in all samples (so not just measuring the amount of receptor – actually measures phosphorylation)

Answer 41

Cellular responses can be monitored in a number of ways, e.g. by quantifying DNA synthesis (if measuring the effect of proliferation) or by direct microscopic observation of cellular behaviour. Ex. by adding GF to sample and observe cell behavior

Answer 42

1. Proximity induced by ligands being a dimer. When each monomer binds to each receptor, it cross-links the receptors, thus also forming a dimer But monomeric ligands can also dimerize RTKs eg. EGF binds to each monomeric EGFR, causes conformational changes to receptor where dimerization arm is exposed, promoting EGFR dimerization. 2. Some receptors = already a dimer, but TKD is far from each other. Binding of ligand brings the TKD domain close to each other. eg. Insulin receptor – a covalent, disulphide-linked dimer * Each monomer = made from ⍺ and β subunit from same gene product that underwent PT proteolysis into separate polypeptide chains, but remain covalently linked via disulphide bond * Binding of insulin brings TKD of insulin receptor close to each other 3. Some RTKs are non-covalently associated in the absence of ligand (ex. DDRs) 4. Some RTKs require higher-order clustering for full activation (ex. DDRs) 5. Some RTKs require co-receptors eg. FGFRs and Heparan sulphate o stretch of repeating disaccharides w/ lot of sulphate on it o essential co-receptor for fibroblast growth factor – without it, no signal. co-receptor gives the cell another mechanism to control the signaling outcome

Answer 43

* For transphosphorylation, thus activation, to occur, 2 kinase domains w/ activation loop in the active conformation have to meet * more likely if the kinase domains are in close proximity, thus dimerization or induced conf change by ligand is necessary. If receptors are far apart, activation = unlikely * Once each receptor is P in the activation loop, the kinase domain is locked in the active conformation (until the P groups are removed by a phosphatase). * Auto-phosphorylation of the activation loop is the first step in the activation of most RTKs.

Answer 44

* Each IRK can be in inactive/ active conformation (exist in dynamic equilibrium) * unP activation loop is predominantly in the inactive conformation (eq prefers inactive conf.) inactive conformation: IRK is autoinhibited by activation loop, w/ Tyr1162 adopting a pseudosubstrate conformation, thus blocking substrate (ATP) binding. tyrosines in the activation loop are unable to be P because they are tucked away. active conformation: activation loop no longer blocks the active site. 3 Tyr residues can then be P and substrate Tyr can occupy the former position of Tyr1162.

Answer 45

* EGFR tyrosine kinase is activated by an allosteric mechanism * inactive form, activation loop of both EGFR monomer blocks the active site * Upon dimerization by EGF binding, "activator" kinase domain in the asymmetric EGFR dimer allosterically activates the "receiver" kinase * causes conf. change of C-helix in the N-lobe of the receiver kinase, pushing the activation loop out of the active site of the receiver kinase * Receiver kinase then autoP the C-terminal tails of the EGFR dimer

Answer 46

P tyrosine residues in the CTD tail act as docking sites for a variety of intracellular signalling proteins

Answer 47

* Phospholipase C-γ (PLC-γ) * Phosphoinositide 3-kinase (PI 3-kinase) P lipid head groups, which become docking sites for effectors. * Non-enzymatic adaptors (Grb2) Ultimately lead to activation of small GTPase Ras and downstream signalling (MAP kinase pathway) – act as mediators that relay signal from the receptor to enzymes

Answer 48

SH2 (and PTB) domains that recognise phosphotyrosines * human genome encodes ~100 SH2 (Src homology region 2) domains * Has a conserved pTyr pocket – a deep pocket with an arginine at the bottom to recognize charged Tyr & a specificity pocket for the residue

Answer 49

* All have SH2 domain for pTyr recognition and other domains for its distinct funtions * modular architecture allows proteins to have multiple interaction domains w/ different properties in the same molecule, thus enhancing specificity and allows the formation of multiprotein signalling complexes. SH2 Phospho-tyrosine binding SH3 Poly-proline binding PTB Phospho-tyrosine binding PH Phospho-inositide binding C2 Membrane binding

Answer 50

> Inactive Src is autoinhibited allosterically by an intramolecular interaction involving SH2 and SH3 domains. SH2 domain is inhibited by interacting with pTyr in CTD >Src activation involves the deP of a regulatory pTyr site in the C-terminal tail, dislodging SH2 towards the back of the active site - Via allosteric mechanism, this causes a conf. change of C helix from up to down conformation, which in turn causes the activation loop to be released and P, allowing access to the active site phosphorylation DOES NOT always lead to activation

Answer 51

Rous sarcoma virus -virus that induces tumor upon infection * Encodes viral oncogene v-src, truncated version of a normal cellular tyrosine kinase (the proto-oncogene c-src) which does not contain the CTD tail. SH2 domain is not inhibited by interaction with pTyr in CTD, so v-src is always active

Answer 52

* MAPK = Mitogen-activated protein kinase * Mitogen = polypeptide that signals cell to enter cell cycle and divide * central to the regulation of cell growth and proliferation. *deregulated in many types of cancer.

Answer 53

small monomeric GTPase * switched on when ATP is bound or switched off when ADP is bound * Contains switch helix, which changes most between the GDP- and GTP-bound states. In active Ras, the switch helix interacts with Ras effectors.

Answer 54

anchored to cell membranes via PTMs * It is a doubly lapidated molecule – (underwent lipidation) to have 2 cysteines near the C terminus * 1 has a farnesyl group (isoprene derived lipid) * 1 has palmitic acid (saturated fatty acids)

Answer 55

small GTPase * Relays signals from surface receptors to the cytoskeleton – important for regulating cell shape * regulate actin dynamics * Depending on which Rho is activated, (Rho, Rac, or CDC42), will result in diff cytoskeletal changes o Rho = stress fibers o Rac = lamellipodia o CDC42 = Filopodia

Answer 56

via Grb2 and Sos Grb2 * a non-enzymatic adaptor molecule that has an SH2 domain, allowing it to bind docking site of RTK * also has SH3 domain that binds polyproline on Sos, recruiting it to the membrane Sos = GEF of Ras (exchanges ADP to ATP) * Sos is near the membrane where Ras is located, it can activate Ras

Answer 57

Ras-GTP allosterically activates Raf – a MAP KKK Raf P Mek, a MAP KK Mek P Erk, a MAP K (all = serine-threonine kinases) Erk then P diff. downstream molecules which cause diff. downstream changes In humans, there are: 16 members of MAP KKK family, 9 members of MAPKK family, 14 members of MAPK family

Answer 58

> use scaffold proteins to physically organize MAP kinase modules > Raf, Mek, Erk held together by KSR > Diff. MAP kinase modules use diff. scaffold proteins, respond to diff. signals, leading to diff. outputs >scaffolding results in loss of massive signal amplification (because 1 kinase can only P 1 kinase) which is a trade-off with specificity

Answer 59

1. slow pathway = gene expression * Active Erk translocate to nucleus to P a TCF (TF) * Active Erk also P another protein kinase p90RSK which also translocate to nucleus to P another transcription factor SRF Erk P 2 transcription factors – both directly and in directly * 2 TF must come together at the SRE cis-regulatory element to lead to transcription of c-fos gene, an immediate early gene. * c-fos protein produced = unstable 2. fast pathway = phosphorylate c-fos protein= stable * sustained input is necessary for c-fos to be stable. stable c-fos protein can combine w/ c-jun to form TF AP-1. AP-1-dependent genes result in cell growth, proliferation, and differentiation.

Answer 60

1. Fast response - P Sos (Ras-GEF) which breaks Grb2-Sos interaction, directly switching off initial signal 2. Slow response - upregulates expression of a MAPK phosphatase (MKP) that deP Erk, preventing downstream activation by Erk

Answer 61

Ras association with GAP results in ATP hydrolysis, switching it to inactive form

Answer 62

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 63

* used by cytokines and hormones: interferon α and γ, erythropoietin, prolactin, and growth hormone. * Recognized by cytokine receptors * Receptor activation similar to RTKs, except that the kinase is a separate protein that is permanently associated with the receptor 1.Upon signal recognition, receptor = dimerize 2. associated JAK to be activated by auto-transphosphorylation 3. active JAK then P the receptor 4. STAT1&2 (signal transducers and activators of transcription) – have SH2 domain. bind to pTyr of receptor 5. STATs are then activated by P by JAK 6. active STAT directly acts as TF to activate gene transcription

Answer 64

* TGFβ superfamily provide important signals in development and many other processes (ex. wound healing). * Recognized by transforming growth factor-β receptors (type I and II) 1. Upon signal recognition, type II receptors are ser/thr kinases that P the type I receptors 2. Type I receptors in turn P the Smad signalling mediators 3. Smad can then directly act as TF that result in gene transcription

Answer 65

STATs and SMADs provide fast tracks to the nucleus * Diff. from RTK pathway that requires multiple steps, thus multiple points of regulation, before gene transcription

Answer 66

* PLC-γ contains SH2 domains and is recruited to the activated RTK * Cleaves PIP2 bond that result in diacylglycerol and the release of IP3 * IP3 opens Ca2+ ion channels in ER membrane

Answer 67

* The PI3K/Akt pathway regulates cell survival and growth 1. Upon recognition of survival signal, resulting in RTK dimerization and auto-phosphorylation, PI3K is recruited to the membrane & is activated 2. PI3K phosphorylates PIP2 to make PIP3 (at membrane – not released) 3. PIP3 is recognised by pleckstrin homology (PH) domains 4. PH domains are in PDK1 and Akt (kinases) – so both = recruited to PIP3 in membrane 5. PDK1, in cooperation with mTOR, P Akt 6. Active Akt dissociate from PIP3 and P Bad, thus inhibiting it * Bad is originally inhibiting apoptosis inhibitory proteins 7. Results in apoptosis inhibitory proteins becoming active thus inhibiting apoptosis

Answer 68

phosphatase PTEN Removes 3rd phosphate on PIP3 to revert back to PIP2 PTEN is one of the most frequently disrupted tumor suppressors in cancer, leading to increased PI3K/Akt signaling, preventing apoptosis and resulting in unregulated cell growth

Answer 69

1. Dephosphorylation: by protein tyrosine phosphatases (PTPs) 2. Endocytosis (more frequently) * remove receptor from cell surface o BUT signaling can continue in endosomal compartments because upon endocytosis, kinase domain will still face cytoplasm o SO if ligand = present when the receptor was endocytosed, will still have active kinase in cell * Initiated by Ubiquitination of activated EGF receptor by the E3 ligase Cbl

Answer 70

multiple point of intersection o Both can activate Phospholipase C o Arrestin (which binds to GPCR) can also activate MAPK pathway o Some RTK can activate G protein

Answer 71

EGF and NGF both activate the Raf-MEK-Erk pathway in PC12 cells BUT: EGF = transient activation = cell proliferation. NGF = sustained activation = cell differentiation.

Answer 72

1. different feedback loops EGF = negative feedback loop NGF = positive feedback loop = involves PKC * If express PMA to activate PKC in EGF pathway, will also result in sustained signaling & differentiation * If express Gö7874 (inhibitor of PKC) in NGF pathway, will result in transient signaling & proliferation instead 2. different receptor activation kinetics * Transient activation of EGFR is due to downregulation by endocytosis. – resulting in transient signaling of Erk by EGFR o Dynamin inhibitors make activation more sustained = can result in cell differentiation. * NGFR receptor = activated for a long time = more sustained response

Cell Signalling Flashcards

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