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PSC2002 Membrane Transport and Cell Signalling in Health and Disease > L13-26: Cell signalling > Flashcards

L13-26: Cell signalling Flashcards

1
Q

How much of the genome accounts for signalling molecules?

A

10-15%

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2
Q

What are the difficulties of targeting ABC transporters?

A

Physiological roles of ABC transporters
Ubiquitous expression
Transporter redundancy
Dose adjustment
Dose adjustment and monitoring when combining inhibitors with drugs with a narrow therapeutic window

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3
Q

What are the basic features of cell communication?

A

Secreting cell
↓synthesis and release
Chemical signal
↓
Target cell
↓Reception
Secretion, metabolism, contraction, cell growth and excitability etc.

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4
Q

What are the general signal processing pathway steps?

A

Chemical signal
↓
Receptor
↓
Transducer
↓
Amplifier
↓
2nd Messenger
↓
Effectors
↓
Response element
↓
Response

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5
Q

What are the components of a chemical signal in a processing pathway?

A

Pheromones, hormones, local hormones, neurotransmitters and cell surface molecules

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6
Q

What are the components of a receptor in a processing pathway?

A

Ion channel-linked, G-protein linked, tyrosine kinase-linked

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7
Q

What are the components of a transducer in a processing pathway?

A

G-proteins, non-receptor tyrosine kinases

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8
Q

What are the components of an amplifier in a processing pathway?

A

Adenylyl cyclase, PLC

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9
Q

What are the components of a 2nd messenger in a processing pathway?

A

Cyclic AMP, InsP3, IP3, Ca2+, DAG, proteins

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10
Q

What are the components of an effector in a processing pathway?

A

Protein kinases, Ca2+-binding proteins,

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11
Q

What are the components of a response element in a processing pathway?

A

Enzymes, ion channels and TFs

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12
Q

What are the components of a response in a processing pathway?

A

Metabolism, secretion, contraction, excitability, gene transcription and cell growth

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13
Q

What are the basic principles of signal processing?

A

Amplification
Heterogeneity (diversity, multiple forms of components)
Information transducer (decoding)
Dynamics (temporal and spatial aspects)

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14
Q

How does heterogeneity work?

A

Different G-proteins generate different responses

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15
Q

What is an example of information transfer via conformational change?

A

Chemical signal to receptor
Receptor to G-protein
G-protein to amplifier
2nd messenger to protein kinase

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16
Q

What is an example of covalent modification?

A

Phosphorylation (addition of terminal PO34- of ATP to OH group)

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17
Q

Where are most common residues phosphorylated?

A

Serine, Threonine residues

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18
Q

What does phosphorylation do to a compound?

A

Change activity or function

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19
Q

How do cAMP dependent protein kinases work?

A

cAMP binds to PKA inducing conformational change
Causing release and activation of catalytic subunit

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20
Q

What are the differences between type I and II PKAs?

A

Type I: Free in cytosol
Type II: forms stable interaction with AKAPs via R (not free in cytosol) catalytic subunits are not released after

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21
Q

What are neurotransmitters?

A

Chemical messengers
Released from one neurone acting at a close site on another eliciting an effect determined by the specific nature of the receptor

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22
Q

What are neurotransmitters mostly?

A

Amino acids, peptides and monoamines

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23
Q

What are the most common neurotransmitters?

A

Acetylcholine - muscles
Glutamate - excitatory, memory and learning
Dopamine - motivation, pleasure (addiction and love)
Serotonin - emotions, wakefulness and temperature regulation
GABA - inhibitory
ATP - neuronal/glial communication, pain regulation

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24
Q

What are the differences between ionotropic and metabotropic receptors?

A

Ionotropic: immediate, 1:1, ligand:channel, uses a single messenger, ionic itself
Meabotropic: Slower, 1:multiple channels, branched, can modulate ion channels, cascades to different functions

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25
Q

What is quantal release?

A

It is the release of neurotransmitter from 1 vesicle meaning they are smaller responses
Release from multiple vesicles means there is a larger response

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26
Q

What is the evidence of termination of an action potential from Ach?

A

Persistent Ach application produces persistent current, receptors can desensitise
Q10 = 2.8, not a diffusion dominated process
Voltage dependence of decay (faster at more negative holding potentials)

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27
Q

Who developed the patch clamp technique?

A

Sakmann and Neher

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28
Q

Why was patch clamp technique so important?

A

Opening and closing of membranes could be visualised

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29
Q

What are the properties of nicotinic ACh receptors?

A

At neuromuscular junction and pre and post synaptic cells in ANS
Permeable to Na+, K+ and Ca2+
5 subunits - 2α, 1β, 1σ and 1ε
α binds to ACh

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30
Q

What are the antagonists of nicotinic acetylcholine receptors?

A

α-bungarotoxin and curare

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31
Q

What is Myasthenia Gravis?

A

Autoimmune disease
Production of antibodies against nicotinic ACh receptors
EPP can’t generate muscle stimulation
2nd subset is autoantibodies against muscle specific kinase

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32
Q

What type of receptors are GABAa, GABAc and glycine?

A

Ionotropic receptors

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33
Q

What are the GABAa, GABAc and glycine receptors permeable to?

A

Cl- and HCO3-

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34
Q

Why are the GABAa, GABAc and glycine receptors inhibitory?

A

As the Ecl is close to the Em and below action potential threshold

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35
Q

What structure do GABAa, GABAc and glycine receptors have?

A

Pentameric structures

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36
Q

What type of receptor is GABAb?

A

Metabotropic

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37
Q

How are the GABAa, GABAc and glycine receptors diverse?

A

Different combinations of their subunits

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38
Q

What does the subunit composition dictate?

A

Receptor properties
Cell surface distribution
Dynamic regulation

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39
Q

What are properties of σ subunits?

A

Sensitive, low desensitisation
Mediates tonic GABAergic currents

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40
Q

How are synaptic receptors modulated?

A

Bnzodiazepines
Mediate sedation
Bind at interface of α/γ2 subunits

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41
Q

How are extra synaptic receptors modulated?

A

Barbituates - increase affinity of GABA
Neurosteroids - both positive and negative allosteric modulation
Alcohol - enhances GABA action

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42
Q

What is the affect of barbiturates on GABA receptors?

A

It locks them in an open state (potentiates)

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43
Q

What is the affect of benzodiazepines on GABA receptors?

A

Eases the openings

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44
Q

What is the P2X receptor?

A

An ionotropic receptor using ATP as agonist
Na+, K+ and Ca2+ permeability
Widespread gila and neuronal expression
ATP released in synaptic vesicles
Neuronal-gila and gila-gila communication

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45
Q

What is glutamate?

A

An amino acid excitatory neurotransmitter in vertebrate nervous systems

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46
Q

What are the different types of glutamate receptors?

A

Ionotropic: transient opening allow net influx of cations, generate excitatory current
Metabotropic: Modulate synaptic transmission

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47
Q

What is glutamate most important for?

A

Leaning, memory and various disorders

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48
Q

What are the 3 main glutamate receptors?

A

AMPA, NMDA and kainate (man made)

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49
Q

How are the main glutamate receptors activated?

A

Glutamate and kainate

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50
Q

Why are kainite receptors different to other glutamate receptors?

A

They are only activated by kainate

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51
Q

What are the properties of glutamate receptors?

A

Co-localised at synapses (mediate fast chemical synaptic transmission)
NMDARs, AMPARs and kainateRs both synaptic and extra synaptic
Pre and post-synaptic

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52
Q

Where are glutamate receptors localised?

A

At post synaptic sites mainly (found using fluorescent labelling)

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53
Q

What is the dominant molecule that travels through NMDARs?

A

Ca2+

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54
Q

What is the dominant molecule that travels through AMPARs?

A

Na+

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55
Q

What is the dominant molecule that travels through KARs?

A

Na+

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56
Q

What are the structural properties of glutamate receptors?

A

Multimeric protein complexes - 4 subunits (tetramer)

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57
Q

What are the structural properties of the glutamate receptor subunits?

A

3 transmembrane domains
A re-entrant loop

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58
Q

What are properties of the AMPAR subunits: GluA1-GluA4?

A

~900AAs, 68-73% identity
Two modifications (alternative splicing and RNA editing)
Homomeric and heteromeric channels can form
Heteromeric: in presence of edited GluA2 determines I-V curve and Ca2+ permeability

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59
Q

What is the I-V curve in AMPAR subunits?

A

It is inwardly rectifying - pass less outwards current than inward at equivalent distance from the reversal potential

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60
Q

What is the flip and flop of glutamate receptors?

A

It is a formation of the receptor using alternative splicing (weakens the strength)

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61
Q

How do the slice variants (flip/flow) affect the receptor kinetics?

A

Flow terminated by:
Deactivation - agonist unbinding so closing of channel, removal of transmitter
Desensitisation - channel closes while agonist is bound

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62
Q

How can rate of desensitisation of glutamate receptors be influenced?

A

By the subunit composition

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63
Q

How can the alternative splicing change receptor function?

A

They can differ between prenatal to adult form of flips or flops giving sustained or transient currents

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64
Q

How does subunit composition impact permeability?

A

The different arrangements can filter out ions they do not transport
For example
GluA1 - allows Ca2+ transport
GluA3 - allows Ca2+ transport
But GluA1 + GluA2 - filters Ca2+

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65
Q

How can editing determine permeability of AMPARs?

A

Editing Q/R site determines Ca2+ permeability
GluA2 receptors
Glutamine swapped for arginine by editing
Changes charge making it impermeable

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66
Q

Where are Ca2+ permeable AMPARs found?

A

Bergman glial cells, some hippocampus neurones and auditory neurone etc.

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67
Q

What can editing of AMPARs mean?

A

Can cause implications for plasticity and excitotoxicity

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68
Q

Where does glutamate bind?

A

Between S1 and 2 extracellular domains

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69
Q

What do auxiliary subunits modulate?

A

AMPAR trafficking and gating

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70
Q

Where are kainate receptors located?

A

Mostly around presynaptic area of neurone

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71
Q

Why are kainate receptors harder to study?

A

Because they have a lower conductance than other glutamate receptors

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72
Q

How could β-amyloid plaques build up in Alzheimers?

A

Using the NMDA receptors being blocked with Mg2+ so β-amyloid binds to the binding site forming plaques

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73
Q

What are the neuronal functions of Ca2+?

A

Transduces electrical into chemical signals
Axon and dendritic elaboration and retraction
Synaptic vesicle release
Synaptic plasticity

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74
Q

Why is Ca2+ used lots?

A

It is a common ion
East to construct proteins which bind to Ca2+ and change shape

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75
Q

What is the importance of Ca2+ transients?

A

They have different locations and shapes which get interpreted by presence and sensitivity using sensors

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76
Q

How can extra-synaptic NMDARs modulate synaptic NMDARs?

A

By causing Ca2+ entry once NMDARs are activated which activates CREB (TF) which expresses BDNF (Brain-derived neurotrophic factor) and release phosphorylating TrkB which forms a signalling cascade
All of this is turned off using extra-synaptic NMDA activation which (CREB inactivated) causes cell death pathways and loss of mitochondrial potential

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77
Q

What is bright-field imaging?

A

Limited ability to make out intracellular organelles
Impossible to identify individual proteins/processes
Make them stand out

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78
Q

What is luminescence?

A

It is the emission of light by a substance not resulting from heat

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79
Q

What are the types of photoluminescence?

A

Bio: luciferase - firefly tails, aequorin- jellyfish
Chemi: glow-sticks

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80
Q

What is phosphorescence?

A

Slow emission of light that has been previously absorbed by a substance is slow
Light emission after illumination

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81
Q

What is fluorescence?

A

Emission of light by a substance that has absorbed light fast
Light emission only during illumination

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82
Q

What is auto-fluorescence?

A

It is fluorescence that is naturally occurring

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83
Q

What is fluorescein?

A

It is a universal dye used in engine coolant and opticians eye drops

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84
Q

What is the Jablonski energy diagram?

A

Excitation of electrons to higher energy level, the fluorescence emission occurs when the energy levels drop to ground state

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85
Q

What are the components of an Epi-Fluorescence microscope?

A

Eyepiece, observation tubes tube lens, fluorescence filter cube turret, objective, stage, stage, stage translation control, base, field diaphragm, condenser aperture diaphragm, mercury arc lamp lamp house, frame and focus knob

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86
Q

What is a dichroic mirror?

A

It reflects below a certain wavelength
Transmits above it

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87
Q

What is the dichroic filter block?

A

Excitation filter
Dichroic mirror
Emission filter

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88
Q

How does a confocal microscope work?

A

Focal plane has an out/in focus light then through the lens which has a laser pointing towards the pinhole through the dichroic mirror which is then detected

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89
Q

How can the smaller image be seen?

A

Dependent on the the chemical used and the lateral resolving power (d) the wavelength and numerical aperture of the lens can be used

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90
Q

What are the 3 major ways to overcome the resolution limit?

A

Structured illumination (SIM)
Stimulated emission depletion (STED)
Localisation (STOM & PALM)

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91
Q

What are the properties of STED super resolution imaging?

A

Up to 60nm X-Y resolution
Up to 130nm Z resolution
Fixed samples

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92
Q

What are the problems with fluorescent microscopy?

A

Get the probe on target
Only label the target
Overcome any sample autofluorescence
Phototoxicity - live-cell consideration
Photobleaching - dye resistance

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93
Q

How can dyes target?

A

Small-molecule probes:
dye chemistry and antibodies

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94
Q

How can fluorescent proteins be used to help overcome problems?

A

They can be genetically manipulated to target protein to express a fluorescent tag

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95
Q

How does dye chemistry help targeting?

A

Live-cell imaging applications
Gets through the cell membrane
Only become fluorescent in certain environments
Accumulate in certain organelles
Very easy to use and visualise

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96
Q

What are the problems of dye chemistry?

A

Limited retention time in the cell/organelle
Limited targets
Specificity
Toxicity

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97
Q

What is used for fixation of label?

A

formaldehyde

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98
Q

What is used for permeabilisation for the label?

A

Mild detergent

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99
Q

What is used for blocking the label?

A

Excess non-specific protein

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100
Q

What is the secondary label?

A

Fluorescent

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101
Q

What are the problems with sample labelling?

A

Small-molecule chemical probes - cannot be fixed sometimes, few specifically target individual proteins
Immunofluorescence techniques - difficult with live cells and required permeabilisation

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102
Q

What is the main solution to the fluorescence problems?

A

Fluorescent proteins

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103
Q

How are fluorescent proteins used?

A

They are genetically manipulated into the DNA which expresses the GFP

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104
Q

Why do fluorescent proteins work well?

A

Because they cover most of the visible spectrum
Monitor events in live cells

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105
Q

How are fluorescent proteins visualised?

A

Using confocal microscopy

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106
Q

What are the problems with fluorescent protein microscopy?

A

Fusion constructs - not native, strong promotors ethane signal, transient transfection (expression level), may perturb protein function
Over-expression artifacts - protein found in unexpected areas

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107
Q

What are the problems with live organelle/ protein tracking?

A

Short exposure times
Bleaching issues
Toxicity
Not known if they are active

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108
Q

What is interaction colocalisation?

A

Used to identify cells/organellse that co-express certain protein and also identify the location of proteins

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109
Q

What is FRET imaging?

A

Forster Resonance Energy Transfer
Measures the interaction and location of interaction of two proteins
Typically one structure is labelled with a donor fluorophore the other an accept fluorophore

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110
Q

What are typical FRET dye pairs?

A

CFP (donor) YFP (acceptor)
Fluorescein (donor) Rhodamine (acceptor)

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111
Q

What is the problem with ion imaging?

A

Photobleaching
Difficult to accurately measure Ca2+ concentration

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112
Q

What is ion imaging?

A

Single excitation - single emission
Only fluoresces when bound to Ca2+
Large increase in fluorescence when bound to Ca2+

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113
Q

What is Fura-2 ion imaging?

A

Dual excitation
Only fluoresces when bound
Large increase in fluorescence when bound
Ratiometric - easy to correct
Can be used to accurately measure Ca2+

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114
Q

What is GCaMP -genetic Ca2+ indicator ion imaging?

A

Based on GFP, calmodulin, M13
Single excitation single emission
Only fluoresces when bound
Large increase when bound

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115
Q

What is the main name of the Ca2+ signalling pathway?

A

The phosphoinositide pathway

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116
Q

What are the main molecules that triggers Ca2+ response?

A

Hormones
Growth factors
Neurotransmitters

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117
Q

What are the different responses from intracellular Ca2+ signalling?

A

Ion permeability
Secretion
Contraction
Metabolism
Fertilisation
DNA synthesis
Development

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118
Q

Why is Ca2+ kept low in cells?

A

Can cause cell death if too high

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119
Q

How is intracellular Ca2+ kept low in cells?

A

Ca-ATPase pumps (Plasma membrane Ca2+ ATPase and Sarco-endoplasmic reticulum Ca2+ ATPase)
Na/Ca exchanger
Mitochondria + other organelles
Proteins (+lipid)

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120
Q

What is the approximate concentration of Ca2+ in and outside of cells?

A

Inside ~100nM
Outside ~ 2-3mM

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121
Q

How do Ca2+ OFF mechanisms work?

A

They operate over a range of concentrations
Needed for constant high concentrations

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122
Q

What is the Ca2+ signalling toolkit?

A

Intracellular Ca2+ increases occur when Inositol 1,4,5-triphosphate acts on InsP3 receptors or Ryanodine receptors
Intracellular Ca2+ decreases when OFF mechansims are stimulated like buffering proteins or sensors allow transport out of the cell using SERCA/PMCA

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123
Q

What are the 3 types of Ca2+ signal?

A

Elementary evens
Global Ca2+ wave (intracellular)
Global Ca2+ wave (intercellular)

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124
Q

What are the steps of the phosphoinositide pathway?

A

1) Channel signal induces conformational change at GPCR (7 spanning TMDs)
2) 3 cytoplasmic loops of receptor activates G-protein
3) Stimulates an amplifier PLC
4) PLC acts on membrane phospholipid phosphatidyl inositol 4,5-bisphosphate (PIP2) gives DAG and IP3 both are second messengers
5) DAG acts within lipid bilayer stimulates PKC
6) IP3 diffuses into cytosol acts on ion channels releasing Ca2+
7) Ca2+ signal augmented by Ca2+ induced Ca2+ release (CICR)
8) Ca2+ act through protein kinase
9) Ca2+ use specific binding protein to induce muscle contraction
10) Ca2+ act directly on ion channels to influence excitability

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125
Q

What is phospholipase C-β?

A

It is an amplifier
Causes the formation of DAG and IP3 from PIP2(in the membrane)

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126
Q

Why is IP3 soluble?

A

To find receptors in the cell (inositol)

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127
Q

How do Ca2+ spikes increase?

A

With hormone frequency

128
Q

What is the visualisation of Ca2+?

A

It is in the form of a series of spikes

129
Q

What are Ca2+ waves?

A

They are formed from Ca2+ promoting more Ca2+ release (CICR) which does not diffuse but aim to promote more release of Ca2+ causing a Ca2+ wave

130
Q

What are the 2 components of Ca2+ signals?

A

Temporal (spike)
Spatial (wave)

131
Q

How do the waves and spikes of Ca2+ signals impact the cell?

A

They are both the equivalent of each other:
Wave is spatial correlate of the spike
Spike is the temporal correlate of the wave

132
Q

What would happen if there was no CICR?

A

Too little Ca2+ release:
Sensor proteins stimulate buffer proteins which bind to 98-99% of them
So the rise is buffered meaning there are no spikes and waves

133
Q

How is too little Ca2+ overcome?

A

Regenerative Ca2+ signal is used to saturate the buffers and generate waveband spikes

134
Q

How does Ca2+ stop being released?

A

The CICR halts as it would use too much energy to be constantly pumped out (ATPase pumps)

135
Q

What happens once OFF mechanisms relax?

A

Another spike is produced

136
Q

How can Ca2+ waves be propagated?

A

Using InsP3 and Ryanidine receptors

137
Q

What stimulus is used from the membrane ryanidine receptors?

A

Unknown

138
Q

What is produced stimulate ryanidine receptors directly?

A

Cyclic adenosine diphosphate ribose

139
Q

Are ryanidine receptors sensitive the IP3?

A

NO

140
Q

Where are most InsP3 receptors?

A

Hepatocytes

141
Q

Where are most ryanidine receptors?

A

Cardiac myocytes

142
Q

How are InsP3 and ryanidine receptors regulated?

A

In a biphasic manner
High cytosolic Ca2+ inhibits Ca2+ release after CICR

143
Q

What is used to stimulate InsP3 and Ryanidine receptors?

A

InsP3R: IP3 and Ca2+
RyR: Ca2+ only
Both: ATP binding (NO HYDROLYSIS)

144
Q

How is the Ca2+ spike and wave generated?

A

Initiation through an agonist producing IP3 releasing Ca2+
Amplification using CICR

145
Q

How is the Ca2+ spike and wave terminated?

A

Inhibition of CICR stopping Ca2+ release turning OFF mechanisms on

146
Q

What are elementary Ca2+ release events?

A

Comes and goes without generating a wave which is reduced in space
Called a Ca2+ puff: InsP3R
Also called a Ca2+ spark: RyR

147
Q

What are the spatial and temporal aspects of puff and sparks?

A

Spatial ~3um
Temporal ~200ms

148
Q

What are the hierarchical organisations of intracellular Ca2+ signalling?

A

Fundamental event - blip/quark
Elementary event - puff/spark (abortive waves)
Global event - lots of CICR

149
Q

How are elementary release events physiological signals?

A

Ca2+ release leads to global Ca2+ stimulating contraction in muscle cells
BUT
K+ leaves the cell using Ca2+ activated K+ channel leading to hyper polarisation which relaxes the cell

150
Q

What is the global Ca2+ wave (intercellular)?

A

It is the spread of a Ca2+ wave through a monolayer of cells
Can occur in endothelial cells lining the windpipe

151
Q

How does the wave take place over the monolayer?

A

Using gap junctions which allow the movement of IP3 and Ca2+ so the wave can take place

152
Q

What is the phosphorylation cascade?

A

Ca2+ rises in the cell
↓
Ca2+ calmodulin
↓
Cam kinase II
↓ Phosphorylation
Phosphorylase kinase - P
↓Phosphorylation
Phosphorylase - P

153
Q

How was the phosphorylation cascade found?

A

Enzyme was attached to beads - chromatography
Activating solution of Ca2+, CaM and ATP
poured down column
Deactivating solution of EGTA pouted down which stops activation of glycogen to glucose
Complex falls apart

154
Q

How can the frequency of Ca2+ spikes be decoded?

A

It can help show the different amounts of enzyme (Ca.CaMKII) activity

155
Q

What affect do the different isoforms of Ca2+ signalling receptors have?

A

Different affinities for their activators

156
Q

What forms from the different isoforms are being expressed?

A

Heterogeneity so different isoforms of different receptors all form different pathways which have different affinities for each molecule

157
Q

What is the cardiac specific calcium signal some?

A

Et-1R
PLCβ1(form PIP2 and IP2)
L-type: RyR2
SERCA2a (pump)
PV (parvalbumins)
CaM, TnC (troponinC)

158
Q

What is the T-cell specific calcium signal some?

A

TCR; IL-2R
PLCγ1; PI 3-K
Orai1; IP3R1
SERCA2b
CR (calreticulin)
CAM

159
Q

What are the different steps in the Ca2+ signalling toolkit?

A

Receptors
Transducers
Channels
Pumps
Buffers
Sensors

160
Q

How can the singalsomes be remodelled?

A

Phenotypically and genotypically

161
Q

What is phenotypic remodelling of signalsomes?

A

It is a change from the outside/ external event
e.g. phosphorylation changing activity of a component

162
Q

What is genotypic remodelling?

A

Mutations that cause activity of components (e.g. CF)

163
Q

When does phenotypic remodelling occur in cells under normal conditions?

A

Heart - increased force of contraction with exercise (cAMP phosphorylation of Ca2+ signalling components)
Liver - calcium signalling altered during regeneration

164
Q

What are the main diseases from phenotypic and genotypic remodelling?

A

Phenotypic - Alzheimers disease
Genotypic - Brody’s disease

165
Q

How does phenotypic remodelling occur leading to Alzheimers disease?

A

Extracellular plaque deposits of the β-amyloid peptide disrupt synaptic transmission
β-amyloid oligomers increase Ca2+ entry via the NMDA receptor
Amyloid precursor protein intracellular domain increased Ca2+ release from stores
Abnormal amyloid metabolism results in an up regulation of neuronal Ca2+ signalling to induce an initial decline in memory

166
Q

How does amyloid metabolism lead to more phenotypic remodelling?

A

AICD (precursor protein transcription factor) used to increase RyR and decrease Calbindin (binds to Ca2+ acting as a buffer) producing more Ca2+ for more processing of prion protein so more amyloid oligomers

167
Q

What is the end result of Alzheimers?

A

It is an amyloid-dependent up-regulation of Ca2+ signalling
Causes constant erasure of memory stores (LTD) during the day and night as Ca2+ levels are constantly high

168
Q

What are the potential therapies for Alzheimers disease?

A

Vitamine D3 (Ca2+ off mechanism)

169
Q

How does positive feedback impact Alzheimers?

A

Bidirectional relationship between Ca2+ signalling and amyloidogenic pathway
Amyloid’s increase Ca2+, stimulation of amyloid precursor protein

170
Q

What is Brody disease?

A

A skeletal muscle genetic disorder
Characterised by stiffness and cramp by prolonged Ca2+ elevation and slowing of relaxation
Mutation is SERCA1
SR unable to refill Ca2+; cytosolic Ca2+ remains elevated

171
Q

How can cancer cells cause remodelling?

A

Alter SERCA pump activty
Alter Ca2+ release through InsP3Rs
Alter resting levels Ca2+

172
Q

What is the structure of InsP3R?

A

It is a tetramer (310kDa subunits)
Half pore complex
N-terminal binding domain
Isoforms from 4 alternatively spliced forms of subunits

173
Q

What is the structure of RyR?

A

Plant alkaloid
Tetramer (560kDa subunits)
Big protein
4 TMDs line the pore
N-terminal cytosolic domain

174
Q

What are the 3 genes for RyR?

A

RyR1 - skeletal muscle
RyR2 - cardiac muscle
RyR3 - non-muscle cells

175
Q

What is the cAMP signal pathway?

A

G-protein coupled receptor
↓
Adenylyl cyclase
↓
cAMP
↓
Protein Kinase A
↓
Response from cell

176
Q

How many types of adenylyl cyclase are there?

A

2

177
Q

What are the 2 types of adenylyl cyclase?

A

Type 1: Found in plasma membrane
9 isoforms - activated by Gαs
Activated by plant alkaloid forskolin (by-passes GPCR)
Regulated by cytosolic Ca2+ (airways)
Type 2: Cytosolic enzyme activated increases HCO3- and Ca2+

178
Q

What is the pharmacological agent that activates type 1 adenylyl cyclase?

A

Forskolin

179
Q

Where are type 2 adenylyl cyclase found?

A

In sperm and epithelia

180
Q

What is the structure of adenylyl cyclase?

A

12 TMDs 2NBDs

181
Q

What are the different OFF mechanisms of cAMP?

A

Inhibit cAMP production
Breakdown cAMP
Remove cAMP from cell

182
Q

How does inhibition of cAMP take place?

A

Active the inhibitors G-protein, Gαi, reduces adenylyl cyclase activity, opposing stimulation by Gαs lowering cAMP

183
Q

How does breakdown of cAMP take place?

A

Using phosphodiesterases

184
Q

What are phosphodiesterases?

A

They are enzymes used to break down cAMP as an OFF mechanism

185
Q

What are the properties of phosphodiesterases?

A

11 isoforms
8 breakdown cAMP, other cGMP or both
Expression is tissue specific
Important in ‘shaping’ local cAMP signal (duration, amplification and spatial localisation)

186
Q

How are phosphodiesterases inhibited?

A

By caffeine and PDE inhibitors used to treat disease symptoms

187
Q

What are the clinical uses of PDE3 inhibitors?

A

Cilastazol - peripheral vascular disease (+cAMP - causes relaxation of blood vessels which increases blood flow)
Milrinone - used for failing hearts
(+cAMP - increase HR and inotrophy)

188
Q

What are the clinical uses of PDE4 inhibitor?

A

Roflumilast - used for COPD
(+cAMP - relaxes airway smooth muscle reducing airway obstruction)

189
Q

How is cAMP removed from cells?

A

ABC transporters actively pump cAMP out of the cell
MRPs also affect duration and aptitude of cAMP signal, plus spatial aspects

190
Q

What is the problem with a linear on and off mechanism of cAMP?

A

Different agonists increase cAMP levels but produce distinct responses in same cells
Some physiological agonists produce cAMP dependent responses but do not appear to change global cAMP cells

191
Q

What findings arise from cAMP activation and termination not being linear?

A

cAMP must be highly localised (compartmentalised) to spatially distinct areas inside cells (microdomains), changes in cAMP are agonist specific

192
Q

How is cAMP compartmentalised?

A

GPCRs localised to different regions of the cell
Restrict diffusion of cAMP from plasma membrane (adenylyl cyclase) to cytosol
Target PKA to distinct sites and substrates in cells

193
Q

What are examples of cAMP signalling being compartmentalised?

A

CFTR activity in epithelial cells
Isolated heart cells response to different cAMP agonists

194
Q

What do phosphodiesterases restrict from PM to cytosol?

A

Diffusion

195
Q

What were the phosphodiesterase restricting diffusion CFTR experiments?

A

CFTR activated by GCPR agonist (adenosine) in polarised airway cells
Cell-attached patch used
Experiment 1 + ADO = lots of activity
Experiment 1 + ADO + 8-STP(antagonist) = less activity
Experiment 2 + ADO to bath = little/no activity
Experiment 2 + ADO + Forskolin = lots of activity

196
Q

What is the effect of inhibiting PDE4?

A

It eliminates compartmentalised cAMP signalling in human airways

197
Q

What are the AKAP proteins?

A

A Kinase Anchoring Proteins

198
Q

What are the structures of AKAP proteins?

A

Targeting domain
Interaction sites
2 C subunits
PKA holoenzyme
2RII
Docking domain on enzyme

199
Q

How do PKA and AKAP interact?

A

PKA binds to AKAP via docking domain on RII
Then is targeted to plasma membrane, organelles and sub cellular structures via targeting domain, PKA close to substrates
AKAPs help assemble signalling complexes forming signalling hubs or signalsomes

200
Q

What is the role of AKAPs in activation of CFTR by cAMP?

A

If PKA is present via AKAP it activates CFTR by cAMP alone (+ATP) and block activation by cAMP by preventing PKAII binding to AKAP

201
Q

What is required for Ezrin (AKAP) to target PKA to CFTR?

A

NHERF1 as it contains PDZ1 binding domains CFTR binds and ERM domain that binds Ezrin

202
Q

How is cAMP dynamic measured in living cells?

A

Using genetically encoded fluorescent sensors to study spatiotemporal cAMP dynamics
Use cAMP binding domains from PKA/EPAC attached to fluorescent proteins
Expressed using plasmids

203
Q

What are the 2 experimental approaches to measure cAMP?

A

FRET - cAMP binding domain, 2 fluorescent proteins undergoing FRET, FRET decreases when cAMP rises
Intesity - cAMP binding domain + fluorescent protein tag, rise in cAMP increases fluorescent intensity

204
Q

Why do 2 different GPCR cAMP agonists cause different functional effects in hear cells?

A

Noradrenaline causes increased contractility
Prostaglandin(PGE1) causes no effect on contractility
They have different spatiotemporal changes in cAMP

205
Q

How was the effect of the same receptor but different response investigated?

A

Activation of -adrenergic receptor elevated cAMP to T-tubules and SR
Phosphorylation of key proteins excitation-contraction coupling - LTCCs, phospholamban (SERCA) and RyR2
Activation of prostanoid receptor = phosphorylation of enzymes in metabolism and TFs
Inhibiting PDEs abolished dependent cAMP signalling + PKA phosphorylation
Key roles illustrated

206
Q

How does increased contraction lead to PKA-dependent phosphorylation of Ca2+ signalling components?

A

Components involved: L-type Ca2+ channels (LTCCs)
Phospholamban
Ryanodine type 2 receptors
Enables:
Larger Ca2+ signal generated, + trigger Ca2+ using LTCCs and Ca2+ store via RyR (increased inotropy)
Removes Ca2+ quicker into stores during diastole enable relaxation (lustropy)
Reload Ca2+ stores better help increase inotropy

207
Q

How does PKA increase LTCC activity?

A

PKA phosphorylation increases open state probability of individual LTCCs in sarcolemma
PKA also recruits more LTCCs to sarcolemma

208
Q

How does phenotypic remodelling by cAMP/PKA pathway affect the SERCA pump?

A

PKA phosphorylation and increase Ca2+ causes dissociation of PLB from SERCA
Increased ATP consumption
Increased chronotropy and inotropy

209
Q

What conclusions can be inferred from β-adrenergic stimulation?

A

Leads to enhanced muscle contraction because cAMP/PKA signalling is restricted to sites and substrates directly involved in excitation-contraction coupling
cAMP and Ca2+ signalling interact to maximise contractile response from cardiac cells

210
Q

How is spatial cAMP signalling disrupted in CF airways?

A

FRET studies used to find a decrease in Cl- influx in CF so less interaction with proteins in cAMP signalling meaning it is not as localised

211
Q

What are the protein kinases activated by increased Ca2+?

A

PKC - Ca2+ and/or lipids
Ca2+/Calmodulin-dependent PKs

212
Q

What is the cGMP signalling pathway?

A

GCPRs NOT INVOLVED
generated guanylyl cyclase (GC)
Soluble and plasma membrane GCs
Binds to protein kinase G to activate (2 isoforms)
PKG phosphorylates serine/threonine residues
cGMP broken down by cGMP-dependent PDEs

213
Q

How is cGMP generated?

A

Guanylyl cyclase

214
Q

What are the different types of guanylyl cyclases’?

A

Soluble form - activated by NO
Plasma membrane bound (pGC) - activated by some peptide agonists

215
Q

What are the different treatments using cGMP?

A

Nitric oxide - activates cGMP in VSM leading to vasodilation and fall in BP - drugs often used to treat angina
Viagra - type 5 cGMP PDE inhibitor; rise in cGMP relaxes smooth muscle in some tissues - treat erection problems and pulmonary hypertension
Heat stable enterotoxin - E.coli in intestine - activates cGMP.PKG phosphorylates and activates CFTR - secretory diarrhoea
LPS (endotoxin) - from gram negative bacteria - increase expression iNOS, causing excessive NO production

216
Q

What are the 3 groups of protein kinase C?

A

Conventional (cPKC) - α, βI, βII, γ
Novel (nPKC) - δ, ε, θ, η
Atypical (aPKC) - ζ, ι/λ

217
Q

What is the structure of PKC?

A

Single polypeptide with regulatory and catalytic domains
11 different isoforms, divide into 3 groups (C, N and A)

218
Q

What is the function of PKC?

A

Require phospholipid binding to be active and Ca2+
R domain has pseudo substrate motif - keeps kinase inactivate by occupy substrate binding site

219
Q

What is the activation cycle for PKC?

A

Agonist binds
Ca2+ binds C2 domain; PKC translocates to PM and binds DAG and C1 domain
PS motif disengages from C4 domain, substrates bind and phosphorylate
All forms artificially activated by phorbol esters directly bind to PKC

220
Q

What are the 2 main types of Ca2+/Calmodulin-dependent protein kinases?

A

Show narrow substrate specificities and those with broad substrate specificities

221
Q

What are the functions of CaMKII?

A

Regulate activity of NMDA receptors phosphorylate sites on NR2A and NR2B
Enhances InsP3 formation by inhibiting inositol polyphosphate 5-phosphatase
Frequency decoding of Ca2+ signals and act as molecular switch ing learning and memory
Phosphorylate PLB to control SERCA2 pump, work in synergy with PKA to inhibit PLB effect

222
Q

What are protein phosphatases?

A

Remove phosphate from phosphorylated proteins
4 major classes Ser/Thr:
1, 2A and 2C broad and overlapping substrate specificity
2B (calcineurin) more restricted specificity - requires Ca2+/CaM
Regulated by inhibitory proteins (type I&II)
One major class of tyrosine PPs - cytosolic and membrane bound

223
Q

What are the chemical inhibitors of protein phosphatases?

A

Okadaic acid (OA) - blocks PP1 and PP2A
Cyclosporin A - specific for calcineurin, clinically for immunosuppresion

224
Q

What is Okadaic acid?

A

Toxin produced by dinoflagellates accumulate in marine sponges & shellfish
Causes diarrhetic shellfish poisoning
Symptoms: severe, acte diarrhoea, vomiting and nausea
Caused by:
Increase paracellular permeability
More active CFTR, fluid loss
Inhibits PP1
PP2A completely inhibited

225
Q

What is the role of phosphatase inhibitory proteins?

A

PKA phosphorylates key enzymes leading to breakdown of glycogen
PKA activates PP inhibitory proteins bind and inhibit glycogen breakdown

226
Q

How do different signalling pathways interact?

A

Synergy between cAMP and Ca2+ in skeletal muscle, key role of phosphorylase kinase

227
Q

How does phosphorylase kinase become active?

A

Requires PKA phosphorylation and Ca2+ binding in skeletal muscle
δ subunit = calmodulin, γ = catalytic
PKA phosphorylation of α and β subunits increases Ca2+ sensitivity

228
Q

How is calcium sensitivity increased in PKA?

A

When phosphorylated

229
Q

What are the principles of blood glucose homeostasis?

A

Blood glucose concentration ~5 mM
Total blood volume ~5 L
Total glucose in blood <5g
Daily intake of glucose ~300g
Glucose concentration rises moderately after meal ~5-7 mM
In disease state can rise to 60 mM

230
Q

What are the 2 regulatory hormones of blood glucose?

A

Insulin and glucagon

231
Q

What would blood profiles of insulin and glucagon be like after 75g glucose consumed?

A

Insulin - high so stimulates insulin secretion
Glucagon - initially high but lowers as it is inhibited

232
Q

What is the role of insulin?

A

Secreted when blood glucose is high and causes it to decrease

233
Q

What is the role of glucagon?

A

Secreted when blood glucose is low and causes it to increase

234
Q

When insulin increases what processes are initiated?

A

Muscle
An increase in glucose uptake
Liver
Increase glycogenesis
Decrease gluconeogenesis

235
Q

When glucagon increases what processes are initiated?

A

Liver
Increased glycogenolysis
Increased gluconeogenesis

236
Q

What are the pancreatic islets?

A

They are scattered throughout the pancreas
Make up 1-2% of pancreatic tissue
α, β and δ cells secrete hormones
<0.5 mm diameter

237
Q

What are the contents of α, β and δ cells?

A

α - Glucagon
β - Insulin
δ - Somatostatin

238
Q

What are the regulators of insulin release?

A

Major: Increase glucose
Minor: Increase AAs
Increase neural input
Increase gut hormones
Decrease adrenaline
Decrease somatostatin

239
Q

What are the regulators of glucagon release?

A

Major: Decrease glucose
Decrease insulin
Increase AAs
Minor: Increase neural input
Increase adrenaline
Decrease gut hormones
Increase cortisol

240
Q

What is the sequence of events of insulin production?

A

1 - Nucleus to ER: insulin gene 3 exons, signal peptide, B-chain, C-peptide and A-chain
2 - ER: Preproinsulin - primary peptide
3 - Immature secretory granules: proinsulin signal peptide cleaved and disulphide bridges formed
4 - Mature secretory granules: Mature insulin C-peptide cleaved

241
Q

What is proinsulin?

A

A single chain of 86 AAs

242
Q

What happens in insulin production after formation of disulphide bonds?

A

Residues 31-65 are cleaved forming C-peptide and insulin (51 AAs)

243
Q

How is insulin stored?

A

As a hexatrimeric form complexed with zinc and released by β cells

244
Q

Why does insulin have a short half life?

A

To be able to release more

245
Q

What can be a diagnostic value of type 1 diabetes?

A

C-peptide as it is inactive but stable in the blood stream

246
Q

What is the glucose sensor in insulin secretion?

A

Glucokinase

247
Q

What happens when little glucose is transported?

A

Glucokinase is stimulated causing glycolysis and TCA cycle producing little ATP which allows the KATP channel to stay open so hyperpolarised
Ca2+ channel closed
Low insulin secretion

248
Q

What happens when lots of glucose is transported?

A

Glucokinase stimulated
Glycolysis and TCA
More ATP
K+ stops leaving as channel closes
Membrane depolarised
Ca2+ channel opes so high Ca2+
High insulin secretion

249
Q

What is a key regulator of insulin?

A

Extracellular glucose concentration

250
Q

What other compounds can regulate insulin secretion?

A

Increased by gut hormones (incretins) and amino acids
Decreased by adrenaline

251
Q

Which monogenes cause diabetes?

A

Inactivating glucokinase MODY(young)/PNDM(neonatal)
Activating KCNJ11 PDNM(neonatal)
Activating ABCC8 - SUR1 PDNM(neonatal)

252
Q

Which conversions are most important posprandial(after food)?

A

Liver: conversion glucose to glycogen and triglyceride
Adipose: glucose to triglyceride

253
Q

What is glycogen?

A

It is a branched polymer of glucose
Formed by 1,4 and 1,6 glycosidic bonds
Converted in the liver
Made using glycogen synthase (1,4) and branching enzyme (1,6)
Degraded using debranching enzyme (1,6) and glycogen phosphorylase (1,4)

254
Q

What happens to dietary glucose when stores are full?

A

Forms VLDL (very low density lipoproteins)

255
Q

How are ketones formed?

A

In a fasting state
Used to provide energy for the brain

256
Q

What is the insulin action on the liver?

A

Increased glycogen synthesis
Increased fatty acid synthesis
Increased protein synthesis
Decreased glycogen degradation
Decreased gluconeogenesis

257
Q

What is the insulin action on the muscle?

A

Increased glucose transport (GLUT4)
Increased glycogen synthesis: glycogen synthase(dephosphorylation)
Increased glucose oxidation: pyruvate dehydrogenase (dephosphorylation)
Increased protein synthesis

258
Q

What is the insulin action on adipose tissue?

A

Increased glucose transport (GLUT4) vesicle to PM
Increased triacylglycerol synthesis (+ acetyl-CoA carboxylase and fatty acid synthase)
Decreased triacylglycerol breakdown release of fatty acids (decrease hormone sensitive lipase (HSL) TAG not broken-down)

259
Q

What is TAG role in adipose tissue when insulin is present?

A

TAG uptake is stimulated when very low density lipoprotein and chylomicrons are provided creating TAG and lipoprotein lipase digests into fatty acid and monoacylglycerol which is transported making TAG and being stored in adipose

260
Q

Which enzymes are activated or repressed in liver during covalent modification?

A

Activated: glycogen synthase and Acetyl-CoA carboxylase
Repressed: Phosphorylase

261
Q

Which enzymes are activated or repressed in liver during gene transcription?

A

Activated: Glucokinase, acetyl-CoA carboxylase and fatty acid synthase
Repressed: Glucose-6-phosphase and PEPCK (phosphoenolpyruvate carboxykinase)

262
Q

How is glycogen synthase stimulated?

A

Insulin and glucose/G6P produces regulatory protein phospohtases
Dephosphorylated glycogen synthase causes activation
Insulin also represses glycogen synthase kinase

263
Q

How is glycogen synthase repressed?

A

PKA, PhK, PKC and CAMKK and glycogen synthase kinase which causes ADP to ATP
Phosphorylated glycogen synthase causes repression

264
Q

How is glycogen phosphorylase stimulated?

A

Ph-kiase is phosphorylated using glucagon (cAMP to PKA) which allows ATP to ADP
Phosphorylates glycogen phosphorylase causing stimulation

265
Q

How is glycogen phosphorylate repressed?

A

Protein phosphotases produced by insulin and increase in G6P/glucose
Dephosphorylation of glycogen phosphorylase causes repression

266
Q

What are the different protein domains involved in insulin signalling?

A

PH - Plekstrin homology
PTB - phosphotyrosine binding domain
SH2 - Src Homology 2
SH3 - Src Homology domain

267
Q

What is the PH domain?

A

Pleckstrin homology domain binds to phosphorylated inositol phospholipid in plasma membrane

268
Q

What is the PTB domain?

A

Phosphotyrosine binding domain binds P-Y residues

269
Q

What is the SH2 domain?

A

Src homology domain 2 binds phosphotyrosine surrounded by unique protein sequences

270
Q

What is the SH3 domain?

A

Src homology domain 3 binds specifically proline rich regions

271
Q

What are the properties of the insulin receptor?

A

It is part of the RTK superfamily
Heterotetramer
190kDa glycoprotein
ααββ subunits held by disulphide bonds
α subunit - insulin binding
β subunit - transmembrane; tyrosine kinase (cytoplasmic tail) PTB and SH2 domains

272
Q

How does insulin bind to the α subunits?

A

transphosphorylation of β units
Activation of tyrosine kinase activity
Phosphorylation of IR generates binding sites for proteins with PTB and SH2 domains

273
Q

What is the function of the kinase activation loop on insulin receptor?

A

Activation of tyrosine kinase
Phosphorylation leads to binding site of SH2 domain

274
Q

What is the function of the juxtamembrane domain on insulin receptor?

A

Phosphorylation creates binding site for PTB domain

275
Q

What is the function of the C-terminal domain on insulin receptor?

A

Regulates IR kinase activity and kinase-adaptor interactions

276
Q

What is the function of the Set/Thr residues on insulin receptor?

A

Inhibits receptor kinase activity

277
Q

What is the Shc-SH2 containing protein?

A

From 3 genes A,B and C
Shc comprises: PTB, CH1 and SH2 domains
Shc binds insulin receptor through PTB and SH2
CH1 has tyrosine residues and becomes phosphorylated which binds proteins with SH2 domains
Grb-Sos signals through MAP kinase

278
Q

What is the insulin receptor substrate?

A

Comprises 4 genes: IRS1-4
IRS1 and 2 are essential for insulin biological action in liver, muscle and adipose
IRS contains PH and PTB domains enable binding to phosphorylated insulin receptor

279
Q

What binding domains does IRS contain?

A

PH and PTB domains

280
Q

What happens to the IR and IRS once binding takes place?

A

It becomes phosphorylated on tyrosine by insulin receptor tyrosine kinase activity

281
Q

What is the Grb2 protein?

A

Growth factor receptor bound protein-2

282
Q

What does the Grb2 protein do?

A

Binds phosphorylated IRS/Shc through SH2 the activates and uncovers the SH3 domains which binds SOS (son of seven less) via SH3 domain

283
Q

What is SOS?

A

Son of Sevenless
Acts as GDP/DTP exchange factor (GEF)

284
Q

When is Ras active/inactive?

A

Inactive: Bound to GDP
Active: Exchanged for GTP (using SOS)

285
Q

What happens when Ras-GTP forms?

A

Activates Raf which activates MAPKK (MEK)
MEK activates MAP-kinase (ERK)

286
Q

What is ERK?

A

Drives growth, differentiation and proliferation

287
Q

What factor of IRS makes it useful?

A

IRS1 has 21 potential Y-phosphorylation sites which makes it a docking protein with SH2 domains

288
Q

How many SH2 domains does PI3K have?

A

2

289
Q

How many SH2 domains does Grb2 have?

A

1

290
Q

How many S/T-phosphorylation sites does IRS1 have?

A

30

291
Q

What are the properties of the S/T-phosphorylation sites on IRS1?

A

They are phosphorylated by either components of insulin signalling or stress kinases which are activated by stress/inflammation

292
Q

How does PI3Kinase pathway lead to AKT activation?

A

Insulin binds IR α subunit → activates IR tyrosine kinase → autophosphorylation of tyrosine
Recruitment of IRS to IR → phosphorylation of IRS on tyrosines
Recruitment of proteins to IRS via SH2 domains → formation of PIP3
Recruitment of AKT/PTB and PDK1 by PIP3 to PM
Dual phosphorylation of AKT
Phosphorylation of AKT substrates

293
Q

What is PI3K?

A

Phosphatidylinositol 3-kinase
2 subunit enzyme, 85 and 110 kDa
p110 - catalytic subunit
p85 - regulatory subunit, 2 SH2 domains and one SH3

294
Q

What is the function of PI3K?

A

Adds a phosphate group to 3 position of inositol ring in phasphatidylinositol

295
Q

What happens once PI3K add a phosphate group to phosphatidylinositol?

A

PIP3 forms which remains membrane bound

296
Q

What is PIP3 function in insulin signalling?

A

Acts as a binding site for PH-domain containing proteins

297
Q

What is a factor about PIP3 during insulin signalling?

A

It is undetectable in unstimulated cells and produced transiently to high levels during insulin stimulation

298
Q

What is PIP3 pleiotropic functions?

A

Insulin signalling
Cell proliferation, apoptosis, motility and immune activation

299
Q

How is PIP3 switched off?

A

Using PTEN (phosphatase and tennis homologue)
Mutations in PTEN associated with cancer and other diseases

300
Q

What is AKT?

A

Protein kinase B (PKB)
3 genes α, β and γ

301
Q

What are the 3 domains of AKT?

A

N-terminal PH domains
Central kinase domain (Thr308)
C-terminal hydrophobic regulatory domain

302
Q

How is AKT activated?

A

By phosphorylation on Thr308 (PDK1) and Ser473 (mTORC2)

303
Q

How does PIP3 activate PDK1 and AKT?

A

As they both have PH domains
Locates proteins to cytoplasmic side of the membrane

304
Q

Where do PDK1 and mTORC2 phosphorylate AKT?

A

PDK1 Thr308
mTORC2 Ser473

305
Q

Which substrates does AKT phosphorylate?

A

AS160 (Rab-GAP) - GLUT4 translocation, glucose transport
TSC2/TSC1 > GAP - cell growth
FOXO1, PGC1α and SIK2 - inhibition of transcription of gluconeogenic enzymes
Glycogen synthase (using GSK-3) - glycogen storage

306
Q

How is insulin stimulation of glut4 using AS160?

A

Glut4 - main glucose transporter in skeletal muscle and adipose
Absence of insulin means glut4 is present intracellularly in storage vesicles
Phosphorylation of AS160 by AKT causes inactivation sp glut4 translocation to PM

307
Q

What is AS160?

A

It is a GTPase that negatively regulates translocation by Rab proteins

308
Q

How does AKT cause protein synthesis?

A

mTORC1 regulates protein synthesis
Regulated by TSC1/2
AKT phosphorylate TSC2 leads to inhibition
Rheb-GTP activates mTORC1
mTORC1 activates eIF4E-BP1(eukaryotic translation initiation factor) and S6K1 leading to translation initiation and ribosome biogenesis

309
Q

What does TSC1/2 complex do?

A

Inhibits rheb by converting form GTP to GDP bound state

310
Q

What is Rheb?

A

Ras homologue enriched in brain

311
Q

What is FOXO1?

A

A transcription factor
Induces G6PC and PEPCK (gluconeogenesis)
Recpreses GCK (glucokinase)

312
Q

What effect does AKT have on FOXO1?

A

It increases degradation

313
Q

How does AKT degrade FOXO1?

A

Insulin → phosphorylates FOXO1 → translocation to cytoplasms → degradation by proteasome → no induction of G6PC/PEPCK expression → relieve inhibition of GCK gene → increased GCK expression

314
Q

What is GSK3?

A

Belongs to CMCG family of proline directed kinases
Downstream of PI3K/AKT
Negatively regulated by Der-phosphorylation
GSK-3 substrates - enzymes, TFs, eIF2B, cytoskeletal proteins and IRS1/2 etc

315
Q

What is AKT effect on GSK3?

A

Causes inactivation at position 21 (Ser)

316
Q
A

PSC2002 Membrane Transport and Cell Signalling in Health and Disease (2 decks)

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