Gray - signals, receptors and activation of downstream signalling Flashcards
(167 cards)
1
Q
What is the aim of signalling in unicellular organisms?
A
- to keep own cell viable
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
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
4
Q
What is a tissue?
A
- cells w/ similar origin that might respond in similar way
- come together as organ to perform function
5
Q
What are the 4 basic types of tissue?
A
- endothelial
- nervous
- connective
- muscle
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
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
8
Q
What does the plasma membrane define?
A
- interface between cell and its env
9
Q
What is an important role of membrane assoc proteins in signalling?
A
- many facilitate signal transduction
- membrane composition important in signal transduction
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
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
12
Q
Why are receptors always proteins?
A
- only molecules complex enough
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
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
15
Q
Why do receptors need a v high affinity for signals?
A
- signals at v low conc (approx 10^-8M)
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
17
Q
What does generating an intracellular response usually involve?
A
- release of a 2nd messenger w/in target cell
- eg. Ca2+, cAMP, IP3
18
Q
What distances can ec signalling molecules operate over?
A
- various, from short to long
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
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)
21
Q
What is autocrine signalling?
A
- DIAG*
- target sites are on same cell
- eg. GFs during tumour formation
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
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)
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
25
Are signals involved in other metabolic pathways?
- usually not, but closely related to other biochemical intermeds (eg. AA derivatives)
26
What hydrophobicity and charge do ligands for cell surface receptors usually have?
- often hydrophilic (eg. peptide hormones)
| - or charged (eg. histamines, adrenaline, acetyl choline)
27
What can ligand binding alter?
- 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
28
What diff effects does adrenaline cause in different cells?
2 types:
- muscarinic (GPCR)
- -> heart muscle cell = decreased freq of contraction
- -> salivary gland cell = secretion of saliva
- nicotinic (ion channels)
- -> skeletal muscle cell = contraction
29
How can ligands induce diff responses in diff cells?
- using diff receptors (eg. adrenaline) or same receptor
| - components of cell affects what happens, eg. secretory cell is never able to contract
30
What is an example of a multisubunit ion channel receptor?
- nicotinic ACh receptor
31
How do multisubunit ion channel receptors work?
- 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
32
Where are nicotinic ACh receptors found?
- in plasma membranes of neurons and at NMJs
| - diff no.s and combos of receptor subunits in muscle and neuronal type receptors
33
What ligand binds to nicotinic ACh receptors?
- ACh
| - also nicotine can fit into binding pocket, as similar shape, size and same charge
34
What poison can affect nicotinic ACh receptors, and how does it affect them?
- snake venom α-bungarotoxin protein
| - irreversibly binds neuromuscular receptor, blocking ligand binding, causing paralysis and resp failure
35
How are cells specialised for neuronal signalling?
- to bring membranes v close together
36
What does a neuronal signalling cell look like?
*DIAG*
37
What is the structure of the nicotinic ACh receptors, and how is this alt by binding ACh?
- 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
38
What happens when nicotinic ACh receptors bind ACh?
- 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
39
What is the process by which APs are transmitted, and how does this differ in neuronal and muscular cells?
- 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)
40
What are T-tubules?
- invaginations in membrane w/ VG Ca2+ in
41
In what cells are VG ion channels found?
- nerve and many others
42
How do VG ion channels work?
- 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
43
What components are involved in muscle contraction?
*DIAG*
44
How is muscle contraction induced?
- 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
45
What type of ligands do single pass receptors use?
- peptide ligands
46
What are examples of types of single pass receptors, and what is their structure?
- 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)
47
What is atrial natriuretic peptide (ANP) and what is its role?
- 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
48
How does atrial natriuretic peptide (ANP) perform its role?
- 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
49
How is guanylate cyclase receptor activated?
- dimerises, so able to change shape in membrane
| - cGMP prod by catalytic domain, which activates cGMP-dep protein kinase
50
What is the role of protein kinases?
- add phosphate groups to proteins to change charge and activity
51
In what ways is phosphorylation involved in involved in virtually all signalling pathways?
- 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
52
What is the role of phosphatases?
- remove phosphate groups
53
What are transforming GF β receptors (TGFβ)?
- receptor kinase dimers (in conjunction w/ other receptor like proteins) that phosphorylate SMAD TFs
54
How do TGFβs inhibit cell prolif?
- 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
55
What is an NLS?
- nuclear localisation seq
56
When are TGFβ signalling defects common?
- in cancers
57
What is an eg. of a TGFβ and what is its role?
- bone morphogenic protein (BMP)
| - roles in heart, neural and cartilage dev and postnatal bone formation
58
What medical app could bone morphogenic protein have?
- use in implants to strengthen bone after fracture
59
What are enz linked single-pass receptors and what structure do they have before/after ligand binds?
- 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
60
What are cytokines and some eg.s?
- 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
61
How are cytokine receptors activated?
- 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
62
Why are JAKs named after Janus?
- '2 faced', as linked to receptor and passing on signal so looking in both directions
63
What is a STAT?
- signal transduction and activation of transcrip (TF)
| - ds component
64
What occurs during the JAK-STAT pathway?
- 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
65
What is an SH2 domain?
- 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
66
What is leptin, and its role?
- 'satiety hormone'
- structurally related to interleukins
- act via cytokine receptor and JAK/STAT
- role in reg appetite and removal causes mice to get fat
67
What are receptor Tyr kinases (RTKs)?
- w/ intrinsic enz activity
| - major class of receptors for peptide hormones (eg. GFs or insulin)
68
What does ligand binding cause in RTKs?
- 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
How were many RTKs identified?
- cancer studies
70
Over what distances can RTKs act?
- diff distances
| - paracrine/endocrine/autocrine
71
How can ligand binding to RTKs promote their dimerisation through causing a conformational change?
- 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
What are HERs and how many are in humans?
- human epidermal GF receptors
| - 4, often act as heterodimers
73
How does HER2 differ?
- active loop config (loop always pushed out) and doesn't bind ligand, complexes w/ HER1, 3 or 4
74
What role does HER2 play in cancer?
- gene amplified in 25% breast cancers, increasing signalling via any HER
75
What can be used to treat breast cancer caused by overexpression of HER2?
- herceptin
- a monoclonal antibody
- binds and prevents HER2 dimerisation
76
How are RTKs activated?
- 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
How are RTKs inactivated?
- undergo endocytosis, followed by recycling or degradation
| - endocytosis of HER1 receptor increases 10x on EGF binding and more likely to be degraded
78
How do adapter proteins link activated RTKs to other signalling proteins, using EGF as an eg.?
- 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
How is downstream signalling activated by RTKs?
- 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
What is Ras, and what is its role?
- small monomeric G protein attached to membrane
| - molecular switch (GTPase switch)
81
How is Ras activated and inactivated?
- 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
What role does Ras play in cancers?
- oncogene
| - often dereg in cancers
83
What does Ras often activate?
- Ser/Thr kinase (eg. Raf), which activates cascade of 3 sequentially activating kinases
- MAPK translocates to nucleus and activates TF
84
What are the structural features of GPCRs (G-protein coupled receptors)?
* 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
What similar signalling pathways do GPCRs mediate?
- hormone
- neurotransmitter
- odorant receptors
- rhodopsins
86
How does adrenaline bind to β2-adrenergic receptor?
- 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
Apart from adrenaline, what else can bind to β2 adrenergic receptor, and how does this differ?
- isoprotonal
- agonist of adrenaline --> lower Kd (analogue that binds tighter)
- 4 residues in TM helices 3, 5 and 6 participate in binding
88
How do GPCRs work?
- 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
How do heterotrimeric G-proteins work as GTPase switches?
- 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
In terms of the β2 adrenergic receptor, what is the effector?
- adenylate cyclase
91
What is the process by which GPCRs are activated?
- 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
What is adrenaline and how is it prod?
- hormone secreted by adrenal gland
| - Tyr --> dopamine --> noradrenaline --> adrenaline
93
What effects does adrenaline binding to diff receptor subtypes of β-adrenergic receptors cause ?
- 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
What is the result of adrenaline binding to α2-adrenergic receptors on muscle cells lining blood vessels?
- 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
How are differences in sensitivity between β2 and α1 adrenergic receptors important?
- 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
How does stimulation of β-adrenergic receptors lead to cAMP increase?
- 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
What is the structure of adenylate cyclase, and how is it activated/inhibited?
- large integral membrane prot
- 2 cat domains on cytosolic face of membrane
- activated by interaction w/ Gsα
- inhibited by interaction w/ Giα
98
How does inhibitory G-protein Gi work?
- 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
What happens in downstream signalling of GPCR?
- 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
What is CREB?
- cAMP response element binding factor
| - a TF
101
How are GPCRs inactivated?
- 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
What are the similarities between RTKs and GPCRs?
- both involve G proteins (but diff ones)
- both GTPase switches
- both involve downstream phosphorylation
- both alt transcrip
103
What is an agonist?
- mimics ligand by binding receptor and causing normal response
104
What is an antagonist?
- binds receptor but doesn't activate it = competitive inhibitor
105
What is the structure of β-adrenergic receptor ligands?
- side chain containing NH group determining affinity for receptor
- catechol ring, req for adenylate cyclase activation
106
What are the medical uses of β-adrenergic agonists and antagonists?
- 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
What diseases are assoc w/ G-protein activation?
- cholera
| - whooping cough
108
How is cholera assoc w/ G-protein activation?
- 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
How is whooping cough assoc w/ G-protein activation?
- 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
What is Rhodopsin, and how is it activated?
- 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
How does Rhodopsin function as a GPCR?
- 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
How does rhodopsin induce closing of cation channels in rod cells?
- 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
How does rod cell adapt to diff light levels?
- 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
What is the role of phosphorylated membrane lipids in signalling?
- activatable stores of 2nd messengers
| - initiators of downstream signalling
115
What is the function of phosphatidylinositol (PI)?
- predominant function = membrane structure
| - approx 1% used for signalling
116
What is an eg. of Ras indep linked signalling?
- PI pathway, in EGF receptor
117
How can EGF receptor activate PLC (phospholipase C), and what does PLC do?
- ds of RTK and GPCRs
| - PLC is effector --> PLCγ activated by some RTKs and PLCβ isoform activated by some G-proteins (Gqα)
118
In inositol lipid signalling pathways where are 2nd messengers derived from, and what is the role of PLC?
- 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
Why is ATP called a TRIphosphate, and not a TRISphosphate?
- triphosphate = phosphates on adj carbons
| - trisphosphate = NOT on adj carbons
120
How are 2nd messengers DAG and IP3 gen?
- mod of membrane phospholipids
121
What is the role of 2nd messengers DAG and IP3?
- 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
What is the process by which Gqα couples GPCRs to PLC and causes downstream PLC activation?
- 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
What levels of Ca2+ are generally maintained in cyto, and how?
- v low levels
| - pumped out by ATP powered Ca2+ pumps across membrane
124
What are the diff fates of PIP2?
- hydrolysed by PLS to DAG and IP3
| - or phosphorylated to PIP3, by PI-3 kinase
125
How is PI-3 kinase recruited to cell?
- activated RTK/cytokine receptors recruit it via SH2 proteins
126
What is the role of PIP3?
- important signal
| - recruits PKB to membrane via PH domain
127
What is the role of PI-3 kinase in cell survival, and what opposing process is there?
- 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
What is the role of PIP3 in Dictyostelium cAMP chemotaxis?
- 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
What ligands do cell surface receptors often have?
- hydrophobic mols
| - peptide hormones (eg. insulin) or charged mols (eg. adrenaline)
130
What role do cell surface receptors have?
- affect enz activity --> fast acting and affect gene expression
131
What are 3 eg.s of intracellular signals?
- 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
What is an eg of how intracellular receptors can act as enzs?
- 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
What is the role of the atrial natriuretic factor receptor?
- activates cGMP dep protein kinase (PKG)
134
How does NO cause relaxation of smooth muscle in the ACh response?
* 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
How does ACh result in paradoxical effects in smooth muscle cells?
- if endothelial layer there, ACh causes muscle cell to relax
- if just smooth muscle cell, ACh causes contraction
136
What is the structure of cytosolic guanylate cyclase (GC)?
- heterodimer
| - 2 NO binding sites, 1 w/ higher affinity
137
What happens to guanylate cyclase at low and high NO concs?
- 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
When and where is NO synthesised?
- 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
In what way does NO act via PKG?
- 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
How does viagra work in NO signalling pathway?
- 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
What other NO signalling pathways are there?
- 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
Apart from ED, what can viagra be used to treat?
- can suppress pulmonary hypertension and cardiac hypertrophy (thickening of heart in response to high pressure) --> as increased cGMP levels in target cells
143
What are the characteristics of intracellular receptors for steroid hormones?
- 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
How is the structure of steroid hormones similar?
- all have 3x 6 membered ring and 1x 5 membered ring
145
What is an important precursor of steroid hormones?
- cholesterol
146
What are some eg.s of related lipid soluble signals, that behave in similar way to steroids?
- retinoic acid (reg cell division, limb cell dev)
| - thyroxine (metabolic hormone, rather than changing gene expression)
147
What is the domain structure of the nuclear receptors, and to what extent is this conserved?
* 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
How were seqs of nuclear receptors compared, and what did this show?
- receptor cloning
| - many orphan receptors
149
What does steroid binding to receptor cause, and how does this differ in related receptors?
- 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
How was nuclear translocation of steroid receptors observed experimentally?
- 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
How do the DNA REs that bind nuclear receptors differ between steroids and related receptors?
- 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
How do steroid and related receptors activate transcrip?
- 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
What happens in plants response to phytohormones?
- hormone binds receptor and causes change in protein conformation
- change in enz activity or expression of particular subset of genes
- alt cellular activity
154
How is plant cell RK signalling similar but diff?
- have cAMP, but no evidence used as 2nd messenger
- Ca 2nd messenger in both
- don't have nuclear receptors like steroids
155
What plant processes does auxin reg?
- cell division, growth and differentiation
- apical dominance
- root branching
- gravitropism, phototropism etc.
156
What is apical dominance?
- represses branching
| - cutting off tip stops it and get side branch growth
157
How was auxin transportation demonstrated experimentally?
* DIAG*
- intact coleoptile = curved
- tip removed = no curve
- opaque cap = no curve
- buried in sand w/ tip exposed = curved
158
How does auxin function as a signal?
- 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
What are the components of auxin signalling pathway?
- auxin responsive genes
- auxin RE BPs
- auxin response factors (ARFs) are transcrip activators that bind AuxREs --> turns on expression of auxin reg genes
160
How do Aux proteins reg transcrip?
- unstable short-lived proteins that block transcrip of auxin med genes
- bind to and inhibit ARFs
161
What is auxin receptor TIR1?
- F-box protein involved in targeting proteins for destruction by proteasome
- F-box controls target specificity and auxin allows interaction w/ target
162
What is the proteasome and what is its role?
- 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
What is the process of ubiquitin mediated proteolysis?
- 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
How does ubiquitin-proteasome pathway reg cell cycle and transcrip?
- 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
What is the importance of ubiquitin-proteasome pathway?
- may be as important as phosphorylation in controlling cellular events
- 100s of E3s in euks
166
What is the role of auxin in allowing TIR1 to target Aux repressors for degrad?
- acts as "molecular glue"
- provides hydrophobic binding pocket for protein to bind to
- so can chemically alt specific degrad
167
How does auxin promote interaction between SCF^TIR1 and AUX/IAA?
- 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)
