Lecture 3 - Introduction to signalling and cell surface receptors Flashcards
Paracrine, juxtacrine, and autocrine: what do they involve?
Paracrine - diffusible signal from an adjacent cell
Juxtacrine - signal molecule directly attached to an adjacent cell
Autocrine - signal arises from the responding cell itself (can be positive or negative feedback)
What is the general signalling mechanism?
Extracellular stimulus interacts with receptor
Receptor activates intracellular pathways, which may include the nucleus, cytoplasm, proteins, etc
Intracellular pathways cause altered protein synthesis and/or cytoplasmic machinery
Altered PS/CM causes altered cell activity
Signalling pathway components
- Extracellular stimulus - 100s in higher eukaryotes; mostly chemical; may act at very low concentrations (<10-8M)
- Receptor protein - High affinity for ligand, binding activates a cascade of intracellular events
- Signalling machinery - Multiple molecular changes: relay and amplify information
- Effectors - Produce the response
- ‘Off’ switch - Mechanism for restoring basal activity once stimulation ceases
Cell surface receptors
- Ligand-gated ion channels (nicotinic acetylcholine/GABAₐ receptor)
- G-protein coupled receptors (muscarinic acetylcholine receptors, adrenoreceptors)
- Enzyme-coupled receptors ()PDGF, insulin, and growth hormone receptors
Nicotinic receptor: what is its ligand, what movement does it facilitate, and what does it do?
Ach
Na+ entry into the cell
Induce an action potential in neurones
Enzyme-coupled receptor: what is its ligand, what activity does it facilitate, and what types are there?
Ligand binding activates enzyme activity inside the cell
Enzyme activity may be a feature of the receptor molecule itself, or of a distinct protein with which it is closely associated.
Multiple types - receptor tyrosine kinases, receptor threonine/serine kinases, protease-linked receptors.
Receptor Tyrosine Kinases (RTKs): what do they do, what are some key features, and what do they do?
Membrane enzymes that phosphorylate proteins on the side chains of tyrosine
- Span the membrane once
- Contain a cytoplasmic tyrosine kinase domain (active site) - receptor and enzyme in one
- Most (EGF, PDGF, etc) dimerise due to aid from mobile monomers
- Dimeric structure is essential for activation
Activation of receptor switches on kinase activity – leading to phosphorylation of the receptor and of target proteins on tyrosine residues
Why is RTK dimerisation essential for function?
- Dimerisation brings the active sites of the monomers close together
- The monomers do trans autophosphorylation - each monomer phosphorylates the others’ activation loops
- Tyrosine kinase activation increases significantly - the receptor activates more and more tyrosine residues throughout itself
- Binding proteins are recruited to the phosphotyrosines and may be phosphorylated
Dynamic RTK recruitment: what does it do, how do recruited proteins recognise the RTK, what proteins are typically recruited, and what do they do?
Enhances efficiency - brings pathway components together and may bring enzymes close to their substrates
Recruited protein recognises specific phosphotyrosine residues – amino acid sequence context
Multiple effectors recruited - signal bifurcation (transduce the signal further) occurs
Signalling adaptor proteins: what are they, what do they do, what examples are there, and what do they do?
Not enzymes - no intrinsic activity
- Bind signalling proteins via various interaction domains
- Help assemble multi-protein complexes at activated receptors
IRS-1: major substrate of the Insulin receptor, recruited via PTB domain - its tyrosine phosphorylation recruits further proteins
SHC: has both PTB and SH2 domains - recruited to activated Insulin Receptor
Grb2: recruited to activated EGF receptor and to Y-phosphorylated SHC via SH2 domain
Grb-2: how is it recruited to RTKs, what other domains does it have, and what does it do?
SH2 domain recruits itself to activated RTKs (e.g. EGF receptor)/other Y-phosphorylated proteins
2 SH3 domains - bind proline-rich peptides
Recruit Sos to the membrane, Sos activates ras, a small GTPase
Small GTPases: what are they, what are they activated by, what do they do, and what forms of it exist?
GTP binding proteins – they bind the guanine nucleotides GDP and GTP
Guanine nucleotide exchange factors (GEFs) cause the exchange of GDP for GTP – switching the protein on
Intrinsic GTPase activity of the protein hydrolyses GTP to GDP – switches the protein off (happens slowly; speeded up by GTPase Activating Proteins (GAPs))
Ras (3 isoforms, K, H and N Ras) frequently activated by RTKs
Ras: where is it found, what is it activated by, and how many SOS molecules activate Ras?
Attached to the membrane by a lipid anchor
Activated by Sos (son of sevenless), recruitment of SOS to the membrane (via Grb2) allows it to access Ras; Ras activated: GDP-GTP exchange
1 SOS may activate multiple Ras molecules - amplification
MAPK cascade: what is it, what does it consist of, what does it cause to occur, and is it an example of amplification?
Powerful and ancient signalling pathway: thought to be present in all eukaryotes
Consists of 3 different protein kinases that are activated sequentially:
* When activated, K1 phosphorylates and activates K2
* K2 then phosphorylates K3 in the cascade, again causing its activation
* K3 phosphorylates multiple target proteins
Sequential phosphorylation of kinases in this way = ‘kinase cascade’
Can provide significant signal amplification
‘The MAP kinase cascade’
General format:
Raf activates MEK on its serine/threonine residues, MEK activates ERK1/ERK2 on its tyrosine/threonine residues (dual specificity - normally only either tyrosine or ser/thr activated but MEK can do both)
ERK1/ERK2 will then go on to
What are MAPK, MAP2K, and MAP3K and what examples of them are there?
MAPK - Mitogen-activated kinase (ERK1/2)
MAP2K - Mitogen-activated kinase kinase (MEK)
MAP3K - Mitogen-activated kinase kinase kinase (ERK1/2)
Role of the Membrane in MAPK activation
Ras is associated with the membrane due to its lipid anchor, Ras GTP recruits Raf to the membrane
Membrane targeting is an essential part of activation
MAPK module protein scaffolding
To prevent inappropriate activation of the wrong module, since there are several modules used for different pathways that have some overlapping activators, some kinases are immobilised on protein scaffolds
This increases efficiency by co-localising the correct proteins, but reduces amplification - some freely-diffusible steps still needed in the pathway
PI3K: what is it, what does it do, and what are the reactions involving it?
Phosphoinositide 3-kinase
Phosphorylate headgroup of inositol lipids at position 3 of the inositol ring
Phosphatidylinotisol (4,5) bisphosphate (PI(4,5)P₂) + PI3K -> Phosphatidylinotisol (3,4,5) trisphosphate (PI(3,4,5)P₃)
TL;DR: PIP₂ + PI3K -> PIP₃
PI3K class IA: what are they, what do they do, where do they function, what classes are there, what are the classes activated by, and what subunits do they have?
Phosphoinositide 3-kinase class IA
Phosphorylate headgroup of PI(4,5)P₂, producing PIP₃
Recruited to the membrane where the substrate is located
- Class IA (activated by RTKs/Ras-GTP)
- Class IB (activated by heterotrimeric G proteins/Ras-GTP)
Heterodimers: 1 catalytic and 1 regulatory subunit
Class IA PIP2(?): what are its subunits, how big are its subunits, what do these subunits do, and what is its secondary messenger?
Class IA: 110kDa catalytic subunit (p110 a, b or d) smaller regulatory subunit (p85 / p55).
The regulatory subunit has an SH2 domain that mediates interaction with phosphotyrosines on activated RTK or signalling adaptor protein.
Product (PIP₃) is a second messenger
Secondary messengers: what are they and what key features are there of them?
Small, intracellular, non-protein molecules that rapidly increase during stimulation and allow for signal amplification
- Typically low concentration in unstimulated cells – rapidly destroyed or removed
- May diffuse rapidly.
- Levels drop quickly once stimulation ceases.
- Interact with specific binding proteins – recruitment and/or allosteric activation
- Often promote protein phosphorylation (direct or indirect activation of protein kinases)
PIP3: where is it found, where is it generated, what do downstream binding proteins contain, how is it removed, and in what ways are they removed?
Restricted to the inner surface of the plasma membrane; some mobility in 2 dimensions
Generated at specific locations near sites of receptor activation
Many downstream binding proteins: possess PH or PX domains
Binding proteins recruited to the plasma membrane: a platform for signal organisation
Rapidly removed by dephosphorylation:
* PIP₃ + PTEN -> PI(4,5)P₂
* PIP₃ + SHIP -> PI(3,4)P₂
- PTEN - removes phosphate at position 3
- SHIP - removes phosphate at position 5
PI3 kinases
Akt
A protein kinase activated downstream of PIP3
S/T kinase
Also known as Protein Kinase B (PKB)
First identified as an oncogene: inappropriate activation of PIP3 / Akt pathway in majority of cancers – reduces apoptosis in cancer cells
Contains a PH domain – binds to PIP3 when it is produced in the membrane
Following recruitment to the membrane, Akt is phosphorylated twice to activate it fully (by PDK1, another PIP3 binding protein and mTORC2).
Stays active until dephosphorylated by phosphoprotein phosphatases
Cell signalling: the physiological importance
Essential for survival
Coordination of cells/tissues in multicellular organisms
Stimulation can have powerful effects on cells (and by extension, the whole body), including driving cell division and preventing apoptosis
Need for BALANCE: responses need to be robust, appropriate and optimal; dysfunction of signalling leads to disease
Signaling errors/disease
Loss of sensitivity/lack of activation - cells do not respond even if appropriate (e.g. non insulin-dependent (type 2) diabetes), often due to malfunction or loss of receptor or signalling intermediate
Inappropriate activation - Process activated in the absence of stimulus (e.g. cancer), may be due to overexpression, activating mutations or ‘off switch’ malfunction
Insulin resistance
Signals from insulin receptor bifurcate – it activates multiple pathways including Ras/MAP kinase and PI 3-K
Insulin resistance: selective loss of IRS signalling.
Multiple S/T phosphorylation of IRS. Glucose regulation lost.
Occurs in obesity, sepsis, type 2 diabetes; risk factor for AD, cardiovascular disease, some cancers, pancreatitis
~25% of adults worldwide have ‘metabolic syndrome’ (includes insulin resistance) (Medscape) – nearly 1.3bn people
Over-active RTKs: Gastrointestinal stromal tumours
GIST: Rare tumour (<1% of all GI tumours)
Poor prognosis: insensitive to conventional chemotherapy or radiation
Mutations: increased activity in RTKs:
c-kit (80%)
Platelet derived growth factor receptor A (10%)
Increase in tyrosine phosphorylation and cell division
Imatinib (Gleevec ®):
Imatinib (Gleevec ®):
Specific inhibitor of certain tyrosine kinases (including Kit and PDGFR)
revolutionised GIST treatment
PIP3 and disease
Major role in cell survival – elevation in tumour cells makes them resistant to apoptosis:
> 50% of human cancers have elevated PIP3
PIK3CA gene (encodes for a Class 1A PI3K catalytic subunit) is the most frequently mutated gene in solid tumours – over-activation of the kinase / amplification of wild-type
Isoform specific PI 3-K inhibitors now being used for some advanced breast cancers and types of leukaemia
Many potential drugs withdrawn, often due to toxicity.
Signal termination mechanisms
Important that cells only produce a response when appropriately stimulated.
Phosphorylated proteins or lipids dephosphorylated by phosphatase enzymes.
Second messengers are destroyed or removed
Receptors can desensitize and may be removed from the membrane by endocytosis (although some can still signal from intracellular compartments)
Balance between signalling and termination ensures that responses are optimized.
Impaired signal termination can cause disease.
Small GTPases and disease: Ras and tumours
Small GTPases inactivate themselves by GTP hydrolysis
Mutations reduce the GTPase activity of Ras – loss of ‘off switch’
Frequent in cancer: K-ras is mutated in 30% of human tumours (90% of pancreatic tumours)
GAPs increase GTPase activity of Ras: important for inactivation
Cell-free system – t1/2 1-5h
Inject GTP-ras into oocyte – all GTP hydrolysed in 5min
Ras GAPs recruited by activated RTKs: help to down-regulate signalling; mutations can lead to Ras overactivity
Ras-GAPs and disease: Neurofibromatosis type 1
Multiple neurofibromas (benign growths arising from Schwann cells) develop under the skin and in the nervous system
Predisposition to cancers
Affects ~1 in 3000 people
Caused by mutations in the Ras GAP NEUROFIBROMIN (NF1): reduced GAP activity
Over-active Ras (and consequently MAPK & PI 3-kinase) signalling
PIP3 removal and disease
Reminder: PIP3 removed by lipid phosphatases
SHIP-1: haematopoetic cells. Reduced activity associated with leukaemia
PTEN
Tumour suppressor: reduced activity predisposes to tumours.
