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MBB343 - Biochemical Signalling > Gray - signals, receptors and activation of downstream signalling > Flashcards

Gray - signals, receptors and activation of downstream signalling Flashcards

1
Q

What is the aim of signalling in unicellular organisms?

A
  • to keep own cell viable
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2
Q

How does signalling in unicellular organisms work?

A
  • respond to changes in env (eg. external medium) –> eg. if runs out of phosphate shuts down pathways using it and tries to prod more
  • detected at cell surface
  • info relayed inside cell
  • gene expression enables cell to cope w/ new ec env
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3
Q

What is signalling in multicellular organisms, and why is it needed?

A
  • subset of genes expressed in particular cell type
  • control of expression enables cells (or tissues) to carry out specialised function
  • for coop and coord of cells to ensure correct function of whole org and prevent uncontrolled prolif
  • ec signalling molecules control processes in cells µ - m away
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4
Q

What is a tissue?

A
  • cells w/ similar origin that might respond in similar way

- come together as organ to perform function

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5
Q

What are the 4 basic types of tissue?

A
  • endothelial
  • nervous
  • connective
  • muscle
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6
Q

What is Dictyostelium and how is signalling important for it?

A
  • euk on borderline of uni and multi-cellularity
  • normally single celled
  • but aggregate when challenged (not enough nutrients etc.) into multicellular organism and behaves like one
  • poss due to secreting signals to communicate w/ other cells –> some cells sacrificed for the greater good
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7
Q

How are gap junctions important connections?

A
  • allow signalling molecules (eg. cAMP, Ca2+) to be passed directly between some animal cells
  • areas where plasma membranes are close
  • connexin proteins form ‘gap junction’ tubes controlling passage of small molecules
  • plants have plasmodesmata
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8
Q

What does the plasma membrane define?

A
  • interface between cell and its env
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9
Q

What is an important role of membrane assoc proteins in signalling?

A
  • many facilitate signal transduction

- membrane composition important in signal transduction

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10
Q

How does membrane composition vary due to lipid rafts?

A
  • lipid rafts are more rigid lipid micro-envs on cell surface, due to sat FA tails, lots of GPI-anchored and assoc proteins, so can pack more closely –> can favour specific protein interactions and activate signalling cascades
  • more fluid when unsat kinky tails, glycerol and TM rich proteins present, so cant pack as closely
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11
Q

What is the process of cell-to-cell communication by ec signalling?

A
  • synthesis of signalling molecule by signalling cell
  • release of signal by signalling cell (may have to be processed to be released) and transport to target cell (eg. diffuse, carried in blood)
  • detection of signal by specific receptor protein
  • change in cellular behaviour triggered by receptor signal complex
  • removal of signal to terminate cellular response
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12
Q

Why are receptors always proteins?

A
  • only molecules complex enough
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13
Q

What is the aim of signal transduction in multicellular organisms?

A
  • process by which ec signals bring about their characteristic effects inside cell, as info is converted from 1 form to another
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14
Q

How do receptor proteins exhibit ligand binding and effector specificity?

A
  • ligands (signals/1st messengers), eg. hormones, GFs, neurotransmitters, bind/activate specific receptors, either w/in or on surface of target cells
  • receptor proteins bind to and interact w/ physiologically active substances to relay signals (across membrane or into nucleus)
  • approx 100 - 100,000 receptors per cell
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15
Q

Why do receptors need a v high affinity for signals?

A
  • signals at v low conc (approx 10^-8M)
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16
Q

Once interaction is made, what can receptor-ligand complexes reg?

A
  • cellular metabolism (eg. adrenaline) and enz activity (eg. activating kinase)
  • nuclear activity leading to transcrip of specific genes and activation of TFs
  • cell dev/differentiation/division
  • changes in cytoskeleton
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17
Q

What does generating an intracellular response usually involve?

A
  • release of a 2nd messenger w/in target cell

- eg. Ca2+, cAMP, IP3

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18
Q

What distances can ec signalling molecules operate over?

A
  • various, from short to long
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19
Q

How can ec signalling molecules operate over long distances?

A
  • eg. hormones in blood (ENDOCRINE signalling), transpiration stream
  • diff effects in diff cell types dep on receptors and cellular machinery
  • can reach any cells but only those w/ receptors able to respond
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20
Q

How can ec signalling molecules operate over short distances?

A
  • only affect target cells in close prox
  • paracrine signalling
  • conc that reaches cell could cause variety of responses, or could be all or nothing (ie. need certain amount of receptor occupied to cause response)
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21
Q

What is autocrine signalling?

A
  • DIAG*
  • target sites are on same cell
  • eg. GFs during tumour formation
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22
Q

What is an example of signalling by plasma membrane attached proteins?

A
  • to differentiate cells during development
  • DIAG*
  • delta tells particular cell to become neuron, but don’t want all cells to
  • expresses delta on surface, but doesn’t excrete it, so only seen by adj cells
  • receptor notch binds and undergoes proteolysis, releasing part of protein into cell, goes into nucleus and suppresses expression of delta
  • self reinforcing and inhibiting
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23
Q

What are the 3 major structural classes of cell surface receptors?

A
  • multi subunit receptors (ion channels)
  • 7 pass receptors (GPCR)
  • single pass receptors (TGF, RTK, cytokine)
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24
Q

What makes a good signal?

A
  • unique enough to relay defined signal and only be detected by correct receptors
  • usually small enough to travel easily
  • synthesised, alt or released quickly to be switched on rapidly
  • degraded or re-sequestered quickly to cease signalling
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25
Q

Are signals involved in other metabolic pathways?

A
  • usually not, but closely related to other biochemical intermeds (eg. AA derivatives)
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26
Q

What hydrophobicity and charge do ligands for cell surface receptors usually have?

A
  • often hydrophilic (eg. peptide hormones)

- or charged (eg. histamines, adrenaline, acetyl choline)

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27
Q

What can ligand binding alter?

A
  • membrane pot –> change ion channel receptor, changing charge across membrane
  • protein kinase activity –> directly or indirectly
  • cytosolic conc of 2nd messengers (eg. Ca2+, cAMP, cGMP, IP3
  • overall affect enz activity and/or gene expression and reasonably fast acting
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28
Q

What diff effects does adrenaline cause in different cells?

A

2 types:

  • muscarinic (GPCR)
  • -> heart muscle cell = decreased freq of contraction
  • -> salivary gland cell = secretion of saliva
  • nicotinic (ion channels)
  • -> skeletal muscle cell = contraction
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29
Q

How can ligands induce diff responses in diff cells?

A
  • using diff receptors (eg. adrenaline) or same receptor

- components of cell affects what happens, eg. secretory cell is never able to contract

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30
Q

What is an example of a multisubunit ion channel receptor?

A
  • nicotinic ACh receptor
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31
Q

How do multisubunit ion channel receptors work?

A
  • ligand binding changes conformation of receptor, so specific ions flow through (can be specific for anion/cation or K+/Ca2+ etc.)
  • altering electric pot across cell membrane
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32
Q

Where are nicotinic ACh receptors found?

A
  • in plasma membranes of neurons and at NMJs

- diff no.s and combos of receptor subunits in muscle and neuronal type receptors

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33
Q

What ligand binds to nicotinic ACh receptors?

A
  • ACh

- also nicotine can fit into binding pocket, as similar shape, size and same charge

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34
Q

What poison can affect nicotinic ACh receptors, and how does it affect them?

A
  • snake venom α-bungarotoxin protein

- irreversibly binds neuromuscular receptor, blocking ligand binding, causing paralysis and resp failure

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35
Q

How are cells specialised for neuronal signalling?

A
  • to bring membranes v close together
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36
Q

What does a neuronal signalling cell look like?

A

DIAG

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37
Q

What is the structure of the nicotinic ACh receptors, and how is this alt by binding ACh?

A
  • pentameric –> 5 related subunits α2β2δ
  • each subunit spans membrane 4x
  • Glu and Asp residues in each subunit, have 2 charged rings to attract cations in
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38
Q

What happens when nicotinic ACh receptors bind ACh?

A
  • binds 2 ec sites, causing conformational change to open channel at centre of pore, by changing helices slightly by rotation of subunits
  • rapid influx of Na+ (selective for Na+)
  • transiently depolarises plasma membrane in region of receptor
  • muscle contraction in response to nerve impulse
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39
Q

What is the process by which APs are transmitted, and how does this differ in neuronal and muscular cells?

A
  • travelling AP depolarises membrane, opening VG Ca2+ channels
  • Ca2+ influx through VG Ca2+ channels
  • triggers release of ACh from stored vesicles
  • ACh binds α subunits of nicotinic ACh receptors on muscle cells
  • Na+ into cell
  • causes change in voltage across membrane, this change in polarity picked up by nearby VG channels
  • so further influx of Na+ = +ve reinforcement of signal
  • signal spreads, so big influx of Na+ and big change in polarity
  • signal gets to T-tubules
  • in neurons this allows Ca2+ into cell, which activates receptors, which release more Ca2+ from ER
  • in muscle cells doesn’t appear to open Ca2+ channels and directly interacts w/ ryanodine receptors to activate
  • ryanodine receptors are in ER (or sarcoplasmic reticulum in muscle cells)
  • Ca2+ release causes muscle contraction (all or nothing)
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40
Q

What are T-tubules?

A
  • invaginations in membrane w/ VG Ca2+ in
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41
Q

In what cells are VG ion channels found?

A
  • nerve and many others
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42
Q

How do VG ion channels work?

A
  • on membrane depolarisation +vely charged voltage sensing helix moves towards ec surface of membrane
  • allows ions in, but opening v transient
  • channel inactivation segment then blocks receptor
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43
Q

What components are involved in muscle contraction?

A

DIAG

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44
Q

How is muscle contraction induced?

A
  • Ca2+ binding causes conformational change in troponin
  • moves tropomyosin so myosin head in contact w/ underlying actin
  • ATP assoc w/ myosin head, causing it to swivel
  • inducing muscle contraction
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45
Q

What type of ligands do single pass receptors use?

A
  • peptide ligands
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46
Q

What are examples of types of single pass receptors, and what is their structure?

A
  • guanylate cyclase receptors –> DIAG ec/TM/catalytic domain
  • TGFβ receptor family
  • receptor tyrosine kinase family (eg. insulin and GF receptors)
  • non catalytic (eg. cytokine receptors)
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47
Q

What is atrial natriuretic peptide (ANP) and what is its role?

A
  • polypeptide hormone secreted by heart muscle cells
  • has guanylate cyclase activity, gen 2nd messenger cGMP to control blood vol by homeostatically reg blood vol and pressure
  • prod as 150bp long and cleaved into smaller molecule to activate
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48
Q

How does atrial natriuretic peptide (ANP) perform its role?

A
  • when bound by vascular muscle cells they relax
  • diuretic = kidneys excrete more water and Na (less water = less pressure)
  • decreases venous return to heart
  • if blood returning no longer at higher pressure then stop making
  • if still is at high pressure keeps making it
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49
Q

How is guanylate cyclase receptor activated?

A
  • dimerises, so able to change shape in membrane

- cGMP prod by catalytic domain, which activates cGMP-dep protein kinase

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50
Q

What is the role of protein kinases?

A
  • add phosphate groups to proteins to change charge and activity
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51
Q

In what ways is phosphorylation involved in involved in virtually all signalling pathways?

A
  • euk kinases phosphorylate Tyr or Ser/Thr residues of prots
  • often activate or deactivate proteins
  • activate many kinases, leading to kinase cascades
  • substrates inc receptors, enzs, MTs, histones, TFs etc.
  • reg phosphatases
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52
Q

What is the role of phosphatases?

A
  • remove phosphate groups
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53
Q

What are transforming GF β receptors (TGFβ)?

A
  • receptor kinase dimers (in conjunction w/ other receptor like proteins) that phosphorylate SMAD TFs
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54
Q

How do TGFβs inhibit cell prolif?

A
  • Ser-Thr kinase activity on intracellular domain, phosphorylates SMAD (intracellular protein) and changes its conformation
  • Ser phosphorylation unmasks NLS and SMAD moves into nucleus
  • ligand binds type II receptor, which recruits and phosphorylates type I receptor
  • type I receptor phosphorylates receptor-reg SMADs (R-SMADs)
  • alts gene expression, inhibiting cell cycle, and therefore prolif
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55
Q

What is an NLS?

A
  • nuclear localisation seq
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56
Q

When are TGFβ signalling defects common?

A
  • in cancers
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57
Q

What is an eg. of a TGFβ and what is its role?

A
  • bone morphogenic protein (BMP)

- roles in heart, neural and cartilage dev and postnatal bone formation

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58
Q

What medical app could bone morphogenic protein have?

A
  • use in implants to strengthen bone after fracture
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59
Q

What are enz linked single-pass receptors and what structure do they have before/after ligand binds?

A
  • receptors w/ no intrinsic enzymatic activity
  • eg. Tyr kinase linked cytokine receptors
  • stable dimeric complex before ligand binds
  • ligand binding stimulates interaction w/ and activation of cytosolic protein kinase, and cross phosphorylation and activation of bound kinase, by bringing dimer closer together
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60
Q

What are cytokines and some eg.s?

A
  • related peptide signals that control diverse range of cellular events
  • interferons –> make cells more resistant to virus infections
  • interleukins –> important for T cell activation
  • prolactin –> induces lactation
  • erythropoietin –> increases prod of RBC precursors
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61
Q

How are cytokine receptors activated?

A
  • kinase and receptor are dimer before ligand bound
  • ligand binding brings receptors and bound JAKs close enough to phosphorylate each other on activation lip
  • conformational change increases kinases activity leading to further phosphorylation of add Tyr residues
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62
Q

Why are JAKs named after Janus?

A
  • ‘2 faced’, as linked to receptor and passing on signal so looking in both directions
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63
Q

What is a STAT?

A
  • signal transduction and activation of transcrip (TF)

- ds component

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64
Q

What occurs during the JAK-STAT pathway?

A
  • STAT binds phosphotyrosines by SH2 domain and is phosphorylated by JAK
  • phosphorylated STAT dissoc and dimerises
  • dimer moved into nucleus, binds DNA and activates transcrip
  • phosphatase inactivates JAK and terminates signal
  • in LT if JAK kept active, ubiquitination and targeted for degrad
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65
Q

What is an SH2 domain?

A
  • Src homology 2 domain
  • conserved domain of approx 100 AAs –> structural features conserved, not necessarily 1° seq
  • binds phosphorylated Tyr
  • over 100 SH2 containing proteins in animals
  • not in yeast or plants
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66
Q

What is leptin, and its role?

A
  • ‘satiety hormone’
  • structurally related to interleukins
  • act via cytokine receptor and JAK/STAT
  • role in reg appetite and removal causes mice to get fat
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67
Q

What are receptor Tyr kinases (RTKs)?

A
  • w/ intrinsic enz activity

- major class of receptors for peptide hormones (eg. GFs or insulin)

68
Q

What does ligand binding cause in RTKs?

A
  • activates Tyr kinase activity
  • stimulates signal transduction cascade
  • by formation of active homo/heterodimer, which cross phosphorylates residues in receptor cytosolic domain to activate intrinsic kinases and forms docking sites for adapter proteins
  • leads to changes in cell physiology and/or gene expression
69
Q

How were many RTKs identified?

A
  • cancer studies
70
Q

Over what distances can RTKs act?

A
  • diff distances

- paracrine/endocrine/autocrine

71
Q

How can ligand binding to RTKs promote their dimerisation through causing a conformational change?

A
  • protruding loops where ligand binding domains interact and EGF (epidermal GF) binding pushes out loops into each other, pulling dimer pair together
  • OR as w/ insulin, already a dimer and activated by ligand binding
72
Q

What are HERs and how many are in humans?

A
  • human epidermal GF receptors

- 4, often act as heterodimers

73
Q

How does HER2 differ?

A
  • active loop config (loop always pushed out) and doesn’t bind ligand, complexes w/ HER1, 3 or 4
74
Q

What role does HER2 play in cancer?

A
  • gene amplified in 25% breast cancers, increasing signalling via any HER
75
Q

What can be used to treat breast cancer caused by overexpression of HER2?

A
  • herceptin
  • a monoclonal antibody
  • binds and prevents HER2 dimerisation
76
Q

How are RTKs activated?

A
  • protein kinase of each receptor monomer initially phosphorylates particular Tyr residues in cytosolic domain of its dimer partner = transphosphorylation (usually 1 kinase dominant and is activator, other is receiver)
  • enhances kinase activity and leads to further phosphorylation of the receptor
  • phosphorylated Tyr residues in activated RTKs act as docking sites for other signal transduction proteins
77
Q

How are RTKs inactivated?

A
  • undergo endocytosis, followed by recycling or degradation

- endocytosis of HER1 receptor increases 10x on EGF binding and more likely to be degraded

78
Q

How do adapter proteins link activated RTKs to other signalling proteins, using EGF as an eg.?

A
  • adapter protein has no enzymatic activity itself –> acts as plug (has SH2 domain)
  • in EGF binds to GEF, so can interact w/ Ras, becomes active, by exchanging GDP for GTP
79
Q

How is downstream signalling activated by RTKs?

A
  • adapter proteins (eg. GRB2) containing SH2 and SH3 domains couple activated receptor to other components of signal transduction pathway
  • SH3 domain (approx 60 AAs) found in proteins that interact w/ Pro-rich regions in other proteins
  • mediate assembly of protein complexes
  • GRB2 binding brings SOS to membrane, so can bind and activate Ras
80
Q

What is Ras, and what is its role?

A
  • small monomeric G protein attached to membrane

- molecular switch (GTPase switch)

81
Q

How is Ras activated and inactivated?

A
  • GDP-Ras = inactive
  • GTP-Ras = active
  • guanine nucleotide exchange factor (GEF), eg. SOS, helps activate Ras
  • Ras has GTPase activity (enhanced by GAP binding) and inactivates itself
82
Q

What role does Ras play in cancers?

A
  • oncogene

- often dereg in cancers

83
Q

What does Ras often activate?

A
  • Ser/Thr kinase (eg. Raf), which activates cascade of 3 sequentially activating kinases
  • MAPK translocates to nucleus and activates TF
84
Q

What are the structural features of GPCRs (G-protein coupled receptors)?

A
  • DIAG*
  • 7 TM α-helices –> structurally similar
  • cytosolic hydrophobic loop (C3) interacts w/ coupled G-protein –> structures of helices changed slightly by binding C3
85
Q

What similar signalling pathways do GPCRs mediate?

A
  • hormone
  • neurotransmitter
  • odorant receptors
  • rhodopsins
86
Q

How does adrenaline bind to β2-adrenergic receptor?

A
  • helices 3, 5 and 6 contrib to ligand specificity (mutation signif decreases binding)
  • on binding, 5 and 6 thought to move relative to one another
  • alt conformation of C3 –> C3 structure determines specificity of G protein interaction
  • allows C3 to bind and activate transducing G protein (Gs = stimulatory or Gi = inhibitory)
87
Q

Apart from adrenaline, what else can bind to β2 adrenergic receptor, and how does this differ?

A
  • isoprotonal
  • agonist of adrenaline –> lower Kd (analogue that binds tighter)
  • 4 residues in TM helices 3, 5 and 6 participate in binding
88
Q

How do GPCRs work?

A
  • ligand binding causes conformational change in GPCR, passed to coupled G-protein
  • GDP replaced by GTP
  • Gα-GTP dissoc from Gβγ (Gβ and Gγ hold in off position)
  • G-protein activates (or inhibits) an enz that gens a 2nd messenger or modulates an ion channel
89
Q

How do heterotrimeric G-proteins work as GTPase switches?

A
  • 3 subunits = α, β, γ
  • Gsα alts between:
  • -> OFF = GDP bound
  • -> ON = GTP bound
  • when on dissoc from receptor and shuffles along membrane to activate effector (membrane protein)
  • GTP rapidly hydrolysed to GDP and switch turns itself off again –> Gsα subunit reassoc w/ βγ and effector is inactivated
90
Q

In terms of the β2 adrenergic receptor, what is the effector?

A
  • adenylate cyclase
91
Q

What is the process by which GPCRs are activated?

A
  • binding of hormone induces conformational change in receptor
  • activated receptor binds to Gα subunit
  • activated receptor causes conformational change in Gα, triggering dissoc of GDP
  • binding of GTP to Gα triggers dissoc of Gα from receptor and from Gβγ
  • hormone dissocs from receptor and Gα binds to effector, activating it
  • hydrolysis of GTP to GDP causes Gα to dissoc from effector and reassoc w/ Gβγ
    ………. and repeats
92
Q

What is adrenaline and how is it prod?

A
  • hormone secreted by adrenal gland

- Tyr –> dopamine –> noradrenaline –> adrenaline

93
Q

What effects does adrenaline binding to diff receptor subtypes of β-adrenergic receptors cause ?

A
  • cardiac muscle cells increase contraction rate and increase blood supply to tissues (β1)
  • hepatic and adipose cells trigger release of glucose and FAs (β2)
  • smooth muscle cells of intestine and bladder relax (β2)
94
Q

What is the result of adrenaline binding to α2-adrenergic receptors on muscle cells lining blood vessels?

A
  • vasoconstriction cuts off circulation to skin, kidneys and intestine
  • to supply energy for rapid movement of major locomotor muscles in response to bodily stress –> fight, flight, frolic
95
Q

How are differences in sensitivity between β2 and α1 adrenergic receptors important?

A
  • β2 = vasodilation, more sensitive, less of them, so more binding at low adrenaline levels
  • α1 = vasoconstriction, less sensitive, more of them, so more binding at high adrenaline levels
96
Q

How does stimulation of β-adrenergic receptors lead to cAMP increase?

A
  • adrenaline binding activates G-protein, which activates adenylate cyclase
  • many adrenaline responses mediated by rise in intracellular 2nd messenger cAMP
  • cAMP mods rates of several enz cat reactions (eg. protein kinase A)
  • binding of many hormones to their receptors induces cAMP increase
97
Q

What is the structure of adenylate cyclase, and how is it activated/inhibited?

A
  • large integral membrane prot
  • 2 cat domains on cytosolic face of membrane
  • activated by interaction w/ Gsα
  • inhibited by interaction w/ Giα
98
Q

How does inhibitory G-protein Gi work?

A
  • Giα subunits inhibit activity of their effector protein when in active GTP bound form
  • cAMP levels can be up/down reg by action of diff G-proteins
  • β1 and β2 receptors activate Gs which activates adenylate cyclase
  • α2 receptors activate Gi (same β and γ subunits as Gs, but diff α subunit)
99
Q

What happens in downstream signalling of GPCR?

A
  • elevated cAMP allows protein kinase A (PKA) to dissoc from inactive complex
  • PKA phosphorylates metabolic enzs and CREB
  • DIAG*
  • PKA inactivates myosin light chain kinase in smooth muscle cells
  • PKA activates glycogen breakdown enzs in energy release cells (liver/fat)
100
Q

What is CREB?

A
  • cAMP response element binding factor

- a TF

101
Q

How are GPCRs inactivated?

A
  • PKA phosphorylates receptor, which desensitises it (feedback)
  • BARK (β-adrenergic receptor kinase) phosphorylates ligand bound to β-adrenergic receptors, leading to β-arrestin binding and endocytosis (only phosphorylates receptor if pathway remains on for long time)
102
Q

What are the similarities between RTKs and GPCRs?

A
  • both involve G proteins (but diff ones)
  • both GTPase switches
  • both involve downstream phosphorylation
  • both alt transcrip
103
Q

What is an agonist?

A
  • mimics ligand by binding receptor and causing normal response
104
Q

What is an antagonist?

A
  • binds receptor but doesn’t activate it = competitive inhibitor
105
Q

What is the structure of β-adrenergic receptor ligands?

A
  • side chain containing NH group determining affinity for receptor
  • catechol ring, req for adenylate cyclase activation
106
Q

What are the medical uses of β-adrenergic agonists and antagonists?

A
  • cardiac muscle cell β1 receptors normally increase heart rate on ligand binding
  • beta blockers (eg. practolol) are β1 selective antagonists that slow heart contractions –> little effect on other cells, as β1 receptors only in cardiac muscle cells
  • β2 receptors of muscle cells lining bronchial passages stimulate relaxation –> β2 selective antagonists (eg. terbutaline) used in asthma treatment
107
Q

What diseases are assoc w/ G-protein activation?

A
  • cholera

- whooping cough

108
Q

How is cholera assoc w/ G-protein activation?

A
  • cholera toxin enters cells lining gut and mods Gsα
  • ADP-ribosylated Gsα-GTP activates adenylate cyclase but can’t hydrolyse GTP to GDP
  • enz locked on, cAMP levels increase
  • causes excessive secretion of Na+ and water from blood to intestinal lumen
  • diarrhoea, vomiting and dehydration
109
Q

How is whooping cough assoc w/ G-protein activation?

A
  • pertussis toxin mods Giα in cells lining airways
  • Gi is ADP-ribosylated and no longer inhibits adenylate cyclase
  • cAMP levels rise
  • massive secretion of fluid from affected cells
110
Q

What is Rhodopsin, and how is it activated?

A
  • a GPCR
  • visual pigment in nods and cones of retina
  • made up of retinal chromophore linked to opsin protein
  • chromophore is light activated and photon absorption causes cis to trans isomerisation of retinal
111
Q

How does Rhodopsin function as a GPCR?

A
  • activated rhodopsin interacts w/ G-protein
  • -> GDP dissoc, GTP binds
  • -> Gtα-GTP dissoc from Gβγ to activate phosphodiesterase
  • activated rhodopsin is unstable and dissoc
  • has integral GAP as needs to be turned off v quickly
  • decreased levels in 2nd messenger in cell
  • activation by just 1 photon can activate 100s on transducing mols before dissoc
112
Q

How does rhodopsin induce closing of cation channels in rod cells?

A
  • can often downreg sensitivity
  • activates phosphodiesterase that hydrolyses cGMP to GMP
  • plasma membrane cGMP gated ion channels close, decreasing Ca2+ influx –> at low levels of cGMP more closed, transient hyperpolarisation of membrane to be more -ve inside, stops nerve impulse and ACh release
113
Q

How does rod cell adapt to diff light levels?

A
  • light activated opsin phosphorylated by rhodopsin kinase, decreasing ability to activate Gαt
  • arrestin binds to highly phosphorylated opsin, blocking Gα activation –> at really high levels induces clathrin med endocytosis
  • transducin moves to other parts of cell –> in bright moved away from membrane stacks, so not as accessible to receptor
  • DIAG*
114
Q

What is the role of phosphorylated membrane lipids in signalling?

A
  • activatable stores of 2nd messengers

- initiators of downstream signalling

115
Q

What is the function of phosphatidylinositol (PI)?

A
  • predominant function = membrane structure

- approx 1% used for signalling

116
Q

What is an eg. of Ras indep linked signalling?

A
  • PI pathway, in EGF receptor
117
Q

How can EGF receptor activate PLC (phospholipase C), and what does PLC do?

A
  • ds of RTK and GPCRs

- PLC is effector –> PLCγ activated by some RTKs and PLCβ isoform activated by some G-proteins (Gqα)

118
Q

In inositol lipid signalling pathways where are 2nd messengers derived from, and what is the role of PLC?

A
  • derived from membrane phospholipid, PI- PLC cleaves PI 4, 5 bisphosphate (PIP2) to release inositol 1, 4, 5 (IP3) into cyto and diacylglycerol (DAG) which remains in plasma membrane
119
Q

Why is ATP called a TRIphosphate, and not a TRISphosphate?

A
  • triphosphate = phosphates on adj carbons

- trisphosphate = NOT on adj carbons

120
Q

How are 2nd messengers DAG and IP3 gen?

A
  • mod of membrane phospholipids
121
Q

What is the role of 2nd messengers DAG and IP3?

A
  • IP3 causes increase in intracellular Ca2+ conc, by release from intracellular stores –> induces secretion from salivary cells and smooth muscle contraction
  • DAG together w/ Ca activates PKC, which reg metabolic enzs and TFs by phosphorylation
122
Q

What is the process by which Gqα couples GPCRs to PLC and causes downstream PLC activation?

A
  • once GTP replaces GDP Gα shuffles along membrane and binds PLS –> digests membrane phospholipid to release IP3 into cyto and leave DAG forming hydrophobic patch
  • IP3 activates IP3 gated Ca2+ channel in ER
  • CA2+ activated PKC binds though C1 domain to hydrophobic DAG in membrane –> phosphorylation of substrates ends signal by phosphorylating DAG to phosphatidic
  • depletion of Ca2+ in intracellular stores signals to store operated channels to release more into cell –> get waves of Ca2+ and inactivation of PKC
123
Q

What levels of Ca2+ are generally maintained in cyto, and how?

A
  • v low levels

- pumped out by ATP powered Ca2+ pumps across membrane

124
Q

What are the diff fates of PIP2?

A
  • hydrolysed by PLS to DAG and IP3

- or phosphorylated to PIP3, by PI-3 kinase

125
Q

How is PI-3 kinase recruited to cell?

A
  • activated RTK/cytokine receptors recruit it via SH2 proteins
126
Q

What is the role of PIP3?

A
  • important signal

- recruits PKB to membrane via PH domain

127
Q

What is the role of PI-3 kinase in cell survival, and what opposing process is there?

A
  • PIP3 activated PKB which inhibits apoptosis
  • so activation of PI-3 kinase promotes cell survival
  • PTEN phosphatase removes phosphate and promotes cell death (PIP3 –> PIP2)
128
Q

What is the role of PIP3 in Dictyostelium cAMP chemotaxis?

A
  • phosphatase doesn’t bind when PIP2 –> PIP3
  • phosphatase binds well to PIP2, amplifies PIP3 amount at front of cell, giving directional sensing
  • get grad of chemoattractant
  • no particular localisation of where receptors are
  • cAMP induces slightly higher PI3K activity at front
  • PIP3 destab PTEN membrane interaction
  • PTEN activity higher at back, so PIP3 degraded faster
  • PIP3 helps organise actin cytoskeleton and forms at leading edge and myosin contracts at rear
  • cell polarisation not stable when cAMP grad removed
129
Q

What ligands do cell surface receptors often have?

A
  • hydrophobic mols

- peptide hormones (eg. insulin) or charged mols (eg. adrenaline)

130
Q

What role do cell surface receptors have?

A
  • affect enz activity –> fast acting and affect gene expression
131
Q

What are 3 eg.s of intracellular signals?

A
  • NO directly affects enz activity of receptor
  • steroid hormone receptor complexes affect transcrip of specific genes by binding enhancer regions
  • auxin sends TFs to be degraded
132
Q

What is an eg of how intracellular receptors can act as enzs?

A
  • No dissolved gas binds to soluble guanylate cyclase
  • activated enz cat synthesis of cGMP
  • relaxes smooth muscle surrounding blood vessels
  • blood vessels expand, increasing blood flow
133
Q

What is the role of the atrial natriuretic factor receptor?

A
  • activates cGMP dep protein kinase (PKG)
134
Q

How does NO cause relaxation of smooth muscle in the ACh response?

A
  • DIAG*
  • in ACh response endothelial cells prod Ca2+
  • releasing NO from arginine
  • NO small and diffuses, acting on surrounding cells (paracrine) = smooth muscle cells
  • activates guanylate cyclase to convert GTP –> cGMP, releasing PPi
  • rise in cGMP leads to activation of PKG, causing relaxation of muscle and vasodilation
135
Q

How does ACh result in paradoxical effects in smooth muscle cells?

A
  • if endothelial layer there, ACh causes muscle cell to relax
  • if just smooth muscle cell, ACh causes contraction
136
Q

What is the structure of cytosolic guanylate cyclase (GC)?

A
  • heterodimer

- 2 NO binding sites, 1 w/ higher affinity

137
Q

What happens to guanylate cyclase at low and high NO concs?

A
  • at low NO, binds to higher affinity haem group of soluble GC –> stim low level of cat activity (so can respond quicker when get high conc)
  • at high NO, binds to 2nd site to fully activate GC
138
Q

When and where is NO synthesised?

A
  • in endothelial cells, in response to ACh and subsequent elevation in cytosolic Ca
  • diffuses locally from site of synthesis, as short half life (2-30 secs)
139
Q

In what way does NO act via PKG?

A
  • smooth muscle contraction involves Ca activation of MLCK, phosphorylation and relocation of myosin head
  • PKG inhibits IP3 receptors
  • Ca levels decrease and MLCK inactivated
140
Q

How does viagra work in NO signalling pathway?

A
  • NO release by nerve terminals in penis causes local blood vessel dilation –> erection
  • cGMP quickly hydrolysed back to GMP-specific phosphodiesterase (PDES)
  • viagra is reversible inhibitor of PDES –> used to treat ED
141
Q

What other NO signalling pathways are there?

A
  • oxygen deprivation at high alt –> blood vessels dilate to permit increased blood flow
  • HDL cholesterol stim NO prod –> dilates blood vessels to prevent ischemia and angina
  • defence against bacterial infection
142
Q

Apart from ED, what can viagra be used to treat?

A
  • can suppress pulmonary hypertension and cardiac hypertrophy (thickening of heart in response to high pressure) –> as increased cGMP levels in target cells
143
Q

What are the characteristics of intracellular receptors for steroid hormones?

A
  • lipophilic –> dissolve and move across membrane
  • transported by carrier protein
  • diffuse through membranes
  • effective for hours or days
  • often influence growth and differentiation –> move into nucleus where can reg gene expression
144
Q

How is the structure of steroid hormones similar?

A
  • all have 3x 6 membered ring and 1x 5 membered ring
145
Q

What is an important precursor of steroid hormones?

A
  • cholesterol
146
Q

What are some eg.s of related lipid soluble signals, that behave in similar way to steroids?

A
  • retinoic acid (reg cell division, limb cell dev)

- thyroxine (metabolic hormone, rather than changing gene expression)

147
Q

What is the domain structure of the nuclear receptors, and to what extent is this conserved?

A
  • DIAG*
  • variable cloning (100-500 AAs) –> 0%, no real conservation, determines activation of gene expression
  • DNA binding domain (68 AAs) = Zn finger –> 42-94%
  • ligand binding domain (225-285 AAs) –> 15-57%
148
Q

How were seqs of nuclear receptors compared, and what did this show?

A
  • receptor cloning

- many orphan receptors

149
Q

What does steroid binding to receptor cause, and how does this differ in related receptors?

A
  • steroid = translocation to nucleus –> binds in cyto and then complex moves into nucleus and reg gene expression
  • related = nuclear localised, so ligand goes all the way through to nucleus before binds and activates
150
Q

How was nuclear translocation of steroid receptors observed experimentally?

A
  • cultured animal cells transfected w/ expression vectors
  • fluorescent labelled antibody detects where β-galactoside expressed
  • saw glucocorticoid receptor of ligand binding domain req for movement into nucleus
151
Q

How do the DNA REs that bind nuclear receptors differ between steroids and related receptors?

A
  • steroid REs = 6 bp inverted repeat, sep by 3 bp, receptor binds as homodimer
  • related REs = direct repeat elements, bind heterodimer of nuclear receptor w/ RXR (receptor monomer common to several pathways)
152
Q

How do steroid and related receptors activate transcrip?

A
  • both via histone acetylation
  • cause acetylation of N-ter Gly tails assoc w/ particular genes –> to open up chromatin and allow transcrip
  • deacetylation in absence of ligand –> repressing transcrip
153
Q

What happens in plants response to phytohormones?

A
  • hormone binds receptor and causes change in protein conformation
  • change in enz activity or expression of particular subset of genes
  • alt cellular activity
154
Q

How is plant cell RK signalling similar but diff?

A
  • have cAMP, but no evidence used as 2nd messenger
  • Ca 2nd messenger in both
  • don’t have nuclear receptors like steroids
155
Q

What plant processes does auxin reg?

A
  • cell division, growth and differentiation
  • apical dominance
  • root branching
  • gravitropism, phototropism etc.
156
Q

What is apical dominance?

A
  • represses branching

- cutting off tip stops it and get side branch growth

157
Q

How was auxin transportation demonstrated experimentally?

A
  • DIAG*
  • intact coleoptile = curved
  • tip removed = no curve
  • opaque cap = no curve
  • buried in sand w/ tip exposed = curved
158
Q

How does auxin function as a signal?

A
  • phototropic signal perceived at shoot tip and transported
  • preferentially transported down dark side of shoot
  • promotes cell division and expansion (elongation)
  • PIN membrane proteins important in allowing auxins to get into and out of cells
159
Q

What are the components of auxin signalling pathway?

A
  • auxin responsive genes
  • auxin RE BPs
  • auxin response factors (ARFs) are transcrip activators that bind AuxREs –> turns on expression of auxin reg genes
160
Q

How do Aux proteins reg transcrip?

A
  • unstable short-lived proteins that block transcrip of auxin med genes
  • bind to and inhibit ARFs
161
Q

What is auxin receptor TIR1?

A
  • F-box protein involved in targeting proteins for destruction by proteasome
  • F-box controls target specificity and auxin allows interaction w/ target
162
Q

What is the proteasome and what is its role?

A
  • multi subunit complex that recognises specific proteins for degrad (far more specific than eg. lysosyme)
  • has caps on each end and barrel shaped core w/ proteolytic active sites which chew up proteins pushed in by cap
  • proteins sent here tagged w/ ubiquitin
163
Q

What is the process of ubiquitin mediated proteolysis?

A
  • E1/E2/E3 all enzs involved
  • E1 activated ubiquitin by bonding it to Cys
  • activated ubiquitin transferred to Cys on E2
  • E3 transfers ubiquitin to Cys of specific target protein
  • polyubiquitinated proteins degraded by proteasome
164
Q

How does ubiquitin-proteasome pathway reg cell cycle and transcrip?

A
  • during anaphase cyclin degraded and sent to proteasome
  • cyclin usually represses CDK –> so degrad allows kinase to be active and promotes cell cycle
  • important in all cell types and in plants
  • proteasome also important in reg NFκβ pathway –> involved in interleukin signalling and expression of inflamm genes (for activation inhibitor has to be degrad)
165
Q

What is the importance of ubiquitin-proteasome pathway?

A
  • may be as important as phosphorylation in controlling cellular events
  • 100s of E3s in euks
166
Q

What is the role of auxin in allowing TIR1 to target Aux repressors for degrad?

A
  • acts as “molecular glue”
  • provides hydrophobic binding pocket for protein to bind to
  • so can chemically alt specific degrad
167
Q

How does auxin promote interaction between SCF^TIR1 and AUX/IAA?

A
  • mediate change in binding (doesn’t cause conformational change)
  • SCF^TIR1 targets Aux proteins for destruction –> auxin binding to TIR1 targets Aux proteins to SCF^TIR1, Aux are ubiquitin tagged and degraded, derepressing ARF TFs
  • ARFs reg transcrip of Aux genes
  • restores repression on pathway (in absence of auxin)

MBB343 - Biochemical Signalling (2 decks)

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