Receptor tyrosine kinases Flashcards
How many families of RTKs are there?
16 - each with many individual receptors
What is the relationship between RTKs and their ligands?
Some receptors bind to one or many ligands
Some ligands bind to one or many receptors
What is the affinity of RTKs for their ligands?
Can have high or low affinity
What are the common functions of RTKs?
Proliferation, survival and growth
What affect do RTKs have on the cell cycle?
Promoting effect
How are RTKs usually named?
After their ligand
What varies between RTKs and why?
The extracellular domain - allowing the RTKs to interact with many different types of ligand
What is the structure of the extracellular domain of EGF?
Cystenine rich
What is the structure of the extracellular domain of PDGF?
Immunoglobulin-like domains
What is the same about the structure of ALL RTKs?
- All are enzyme-linked receptors (intracellular domain has kinase activity)
- All have a single transmembrane domain that lacks structure and can ‘snake’ backwards and forwards in the membrane
How is ligand binding to RTKs transduced to the inside of the cell?
1) RTKs in the membrane are not close together
2) Dimerised signalling ligand interacts with 2 RTKs in the membrane
3) This brings the receptors in close proximity to each other and orientates them in the right way so that they can interact perfectly- allowing them to to dimerise
4) Cross phosphorylation of the receptors - resulting in a highly active TK
What does ‘canonical’ mean?
The ‘most common’ way (typical)
What is oligomerisation?
Monomers coming together to form a polymer or a dimer
When do intracellular TK domains have a low activity?
When there is no ligand bound - they can not interact and cant phosphorylate each other at the kinase domain
What 3 things does cross-phosphoylation of the TK cause?
1) Increased activity of the kinase
2) Stabilisation of the receptor in the active state (ligand independant)
3) Causes the kinase to phosphorylate other TYROSINES in the receptor - creating docking site
What are the 2 tools for analysis of the RTK singnalling?
What do they exploit?
Exploit dimerisation:
1) Generation of a dominant negative receptor (mutation in the kinase domain)
2) Constitutive activation of the receptor (over-activation)
What does generating a dominant negative TK domain do?
Why is this method advantageous?
- This receptor blocks the activation of the functional TK receptor as no cross-phosphorylation (which is required for stabilisation) can occur
- Poisons the endogenous form of the receptor
Method is advantageous as do not need to KO the receptor in order to study
How is constitutive activation of the TK receptor achieved?
- Genetically engineer a homodimerisation domain on the extracellular portion of the RTK
- Domain dimerises independantly of the ligand binding domain
What are HSPGs?
Heparan sulphate proteoglycans
Where are HSPGs present?
In the extracellular matrix
What do HSPGs interact with?
The extracellular domain of RTKs
What is the structure of HSPGs?
- Protein core (proteoglycan)
- Long chain of sugars (heparin component) built onto the protein core
What are the 3 forms that the protein core of HSPGs come in and what are they called?
1) Inserted into the membrane - Syndecan
2) Tethered to the extracellular membrane - Glypican
3) Secreted - Periecan
What is the structure of the heparin sugars?
2 saccharide repeats
How can the heparin sugar on HSPGs be modified and what does this do?
Many different ways but especially by sulphation (different levels of sulphation at different positions)
Results in a ‘code’ creating binding sites for specific proteins (ligands and receptors)
What do the sugar chains of HSPGs interact with?
What does this interaction help with?
FGF ligands in the extracellular space
Helps with the formation of a complex with the receptors to allow activation (act as co-receptors)
How does the phosphoylated RTK transduce the signal?
- Cross-phosphorylation of the tyrosines form docking sites
- Docking sites are recognised by intracellular signalling proteins
- These proteins are recruited and held close to the membrane
How do proteins recognise the docking sites on the RTKs?
Proteins that dock have 2 pockets:
- Pocket for the phosphotyrosine
- Pocket for an amino acid side chain
What confers specificity to the protein binding to the docking site on the RTK?
Amino acid side chains which bind to a pocket in the protein
How can the cell distinguish between 2 different RTKs?
Because their docking sites are recognised by different proteins
What is the most common domain that interacts with phosphorylated tyrosines?
SH2 - Src Homology Domain 2
What is the amino acid sequence recognised by SH2?
Glutamatic acid - Glutatmatic acid - Isoleucine
What happens when the PDGF receptor is activated?
It is phosphorylated at 5 different positions by:
- PI 3-kinase (2 sites)
- GAP (1 site)
- PLC-gamma (2 sites)
What 3 components are important in RTK pathways?
PI 3-kinase
GAP (GTPase activating protein)
PLC-gamma
What domains do PI 3-kinase, GAP and PLC-gamma all have in common and what does each domain do?
A SH2 domain - recognise phosphorylated tyrosines
A SH3 domain - mediates protein-protein interactions
What does GAP do?
Activates the RAS/MAP kinase branch
What does PI 3-kinase do?
Activates the ionsitol lipid pathway
What does PLC-gamma do?
Activates the ionsitol lipid pathway
What are the 2 main branches of RTK signalling?
RAS pathway and inositol pathway
What is Ras and what does it do?
A GTPase - binds to GTP (Ras is active) and hydrolyses it into GDP (Ras is inactive)
How is Ras activated by RTK?
1) RTK creates docking sites for GRB2 (has an SH2 domain)
2) Sos (a GEF) binds to the SH3 domain on GRB2 and also to inactive Ras - coupling Ras to the TK
3) Sos promotes the dissociation of GDP from Ras
4) GDP dissociates and GTP binds
5) Sos dissociates
What is Sos?
- A GEF (guanine exchange factor) that binds to SH3 on GRB2 and inactive Ras, to couple Ras to the TK
- Promotes the dissociation of GDP
What does activated Ras activate?
What does this allow?
The Map kinase pathway (signalling cascade)
Allows amplification
What is the MAP kinase pathway?
1) Map kinase kinase kinase (Raf)
2) Map kinase kinase (Mek)
3) Map kinase (Erk)
In the Map kinase pathway, why is it important that the phosphorylation isnt stable?
To allow a transient response - can be easily and quickly shut down
What 4 methods allow the study of signalling in the cell?
1) Visualisation/detection of interactions/signalling
2) Chemical inhibitors (disrupt signalling using antagonists/agonists)
3) Mis-expression/overexpression (constitutively active/ dominant negative forms)
4) Genetic methods (forwards, reverse genetics, mutation analysis, transgeneics)
What are the 2 ways of detecting interactions between proteins?
1) Biochemical methods (in vitro) in columns - based upon physical interactions
2) In vivo: Fret analysis
Describe the fluorescence microscopy camera and the pathway of light
Light source (a specific wavelength):
- Shines into the middle chamber and passes through an excitation filter
- Hits a mirror - reflects the light onto the specimen
Some light passes through specimen but some is absorbed and reemitted at a longer wavelength:
- Hits the dichroic mirror (beam splitter)
- Passes through a barrier filter
What does the excitation filter do in the fluorescence microscopy camera?
Filters the light from the light source to only let through a specific wavelength
What does the dichroic mirror (beam splitter) do in the fluorescence microscopy camera?
Reflect the shorter wavelengths to the sample
Lets the longer wavelengths through to the eye piece
What is pseudo colour and why is it needed?
Colour added to a picture of a sample using computer software
Needed because grey scale cameras are used in fluorescence studies (colour cameras are not as sensitive)
Why is previous study needed before fluorescence studies?
- Must know the wavelengths absorbed and emmitted by the sample
- Need to fit the right filters and beam splitter
What is FRET and what is it used for?
Fluorescence Resonance Energy Transfer
Used to monitor where the proteins are in the cell and follow their movements when signalling is activated
Used to monitor the interaction between 2 different proteins in living cells - can see where in the cell this interaction happens
How is a protein labelled with colour in FRET analysis?
Fluorophore attached to a peptide linker which is attached to a protein of interest
This is translated as one protein
Using FRET, how can you see if 2 proteins interact?
1) Label 2 proteins with different colours (eg. X with blue, Y with green)
2) If these 2 proteins interact, the emission from one protein can activate the other
(blue is excited by violet and emits blue, green is excited by blue and emits green)
3) So, if interact - can excite with violet and see green. If don’t interact - can excite with violet and only see blue
What are the 3 uses of FRET?
1) Study the interactions between 2 different proteins
2) Study conformational changes in a single proteins
3) Show when a protein is cleaved
How does FRET show conformational changes in a single protein?
- Put 2 fluorophores in 2 different parts of the protein
- Conformational change may bring these 2 parts together
How does FRET show cleavage of a protein?
Activity when the flurophores are close together
When the protein is cleaved - proteins drift apart
