Receptor Tyrosine Flashcards
How many families of receptor tyrosine kinases are there ?
What is in each family ?
What is promiscuity ?
What are common functions of RTKs?
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A group of receptors that binds to a specific ligand.
Ligands can interact with multiple different receptors and vice versa.
Stimulate proliferation, cell growth and survival.
What is the general structure of RTKs
Why do the extracellular domains vary
They have a single transmembrane spanning domain.
The ligand binds to the extra cellular domain.
The intercellular domains have kinase activity and catalyse the phosphorylation of proteins.
The variety of the extra cellular domains allows the RTKs to interact with many types of ligand.
PDGF receptor has immunoglobulin domains on the extra cellular domain.
EGF has two cystine rich domains on the extra cellular domain.
Canonical RTK activation
How are docking sites made
The ligand binds to two receptors at once and facilitates receptor dimerisation.
This brings the two receptors in proximity with each other so they can phosphorylate each other.
This increases the activity of the kinase domain and stabilises the receptor in its active state.
It also causes the kinase to phosphorylate other tyrosines in the receptor to create docking sites.
Dominant negative receptors
We create these to study RTK signalling.
This is a normal RTK but it has a mutation in the tyrosine kinase domain so when the ligand binds there is no kinase activity.
It is called dominant negative because the mutant blocks the activity of the functional RTK it is dimerised to.
Constitutive active receptors
Create a RTK that is normal but put a homodimerisation domain on the extra cellular domain to allow the RTKs to dimerise and become activated without a ligand binding.
This means they would be active all the time and if it is responsible for cell proliferation it will cause cells to divide too much.
Heperan sulphate proteoglycan what do they do
What are the three types
What is their basic structure
Interact with the extra cellular domain of RTKs.
One is inserted into the membrane syndecan , one is tethered to the extra cellular part of the cell glypican and the other is completely secreted perlecan.
They all have a protein core and off this comes long sugar chains.
How can HSPGs be modified and what does this allow them to do
How do they act as a co receptor
The sugar chains can be modified by sulphation.
The different modifications allow them to interact with different proteins.
They facilitate the formation of the receptor ligand complex to allow activation of the kinase.
What recognises the docking sites
What pockets do these proteins have and why does it make them specific
Intracellular signalling proteins.
They bind to the phosphorylated tyrosines and are held close to the membrane.
A pocket for binding to phosphotyrosine.
Other pockets for recognising neighbouring amino acids.
The signalling proteins will have many specific pockets for specific docking sites.
What does the SH2 domain do and what pockets does it have
It binds to phosphotyrosine docking sites.
It has a phosphotyrosine pocket and then two glutamic acid pockets and then an isoleucine pocket.
What happens when the PDGF receptor is activated
How are the docking sites named and what do they interact with
What domains do the binding proteins have and why are they important
It is phosphorylated and has five different docking sites.
By which number amino acid they are.
The first two docking sites interact with PI3 kinase. The third site binds to GAP.
The fourth and fifth site binds to phospholipase C.
They have an SH2 and SH3 domain to mediate protein protein interactions.
What does GAP activate
What does PI3 kinase and phospholipase c activate.
What are these pathways
RasMAP kinase branch.
Inositol lipid pathway.
Two main branches in the RTK signalling pathway.
What is Ras
How is it activated and what does it look like when it is.
A group of GTPases that do many functions in the cell.
They are GTP binding proteins.
When they are bound to GTP they are active and when they are bound to GDP they are inactive. This acts as a switch.
When GTP is bound it works like a timer and is only stable for a certain time period.
Ras changes confirmation dramatically depending on whether it is bound to GTP or GDP.
How is the Ras signalling cascade activated
What is SOS and what does it do.
GRB?
What is the rest of the pathway.
Why do we need so many kinases
How can it be shut down
Inactive Ras is tethered to the membrane.
An RTK is activated and forms docking sites for SH2 and SOS which bind to the RTK.
SOS binds to ras and promotes dissociation of GDP from inactive Ras so GTP can now bind.
SOS is a guanine nucleotide exchange factor.
SOS binds to the RTK using GRB2.
Ras will dissociate when it is active and then Ras activates raf. Which activates Mek then erk which are all MAP kinases.
We need so many kinases for amplification of the signal.
The pathway can be shut down quickly because the phosphorylated kinases are unstable so the pathway is rapid and transient.
Methods to study signalling
Could use bead columns to purify proteins or this can be done in vivo using fret.
Chemical inhibitors to disrupt signalling by manipulating a particular component using agonists or antagonists.
Miss expression or over expression to make constructively active or dominant negative forms.
Fluorescent microscopy.
What do fluorescent molecules do
What light is used
What do you need to know before hand
What is pseudo colour
Absorb light of one wavelength and emit light of a longer wavelength.
The microscopes use a very strong light source either a laser or mercury lamp.
You need to know the fluorescent protein you are going to use before so you can choose the filters and dichroic mirror.
Colour cameras are not sensitive so we use grey scale cameras and the picture is recolourised using a computer.
What are fluorescent protein fusions and what do they do
A peptide links a protein and a fluorophore
We can find the protein by shining a light on it.
Let’s us monitor where the proteins are in the cell.
What does FRET stand for and what does it do.
How does it work
Forster resonance energy transfer.
It helps to monitor protein interactions in real time in the cell.
One protein is blue fluorescent and one is green.
If they are interacting they will be very close together so if you shine violet light on them it will excite the blue one and make it emit blue light to the green on and make it emit green light.
If the proteins were not interacting the blue light made would not reach the green protein and the green protein wouldn’t fluoresce.
How can FRET be used to study conformational change
You add a blue fluorescent protein to one end and a green one to the other end of the target protein.
When the ligand and target protein bind it will cause a conformational change that will bring the two ends of the protein together.
So when violet light is shined now it is able to excite the green protein.
How can FRET be used to show when a protein is cleaved
The target protein has a blue and a green fluorescent protein bound and they can make green light.
When it is cleaved the blue and green proteins will be separated and the green light can no longer be produced.
What three families are contained within the TGFB superfamily and what are some examples
BMP like family-
BMPs, GDFs, AMH.
TGFB like family-
TGFB1, 2 and 3. Activins and nodal.
GNDF family-
GDNF, Artemin, neuturin
How does the TGF ligand activate its receptor and what happens next
What would be different if it was BMP or activins
It binds to the TGFBR2 and this will cause the TGFBR1 receptor to join and dimerise.
This will cause phosphorylation.
This will cause phosphorylation of SMAD 2 and 3.
BMP or activins would cause SMAD1 and 5 phosphorylation.
SMAD 4 the binds to the two other SMADS and forms a complex which can act as a TF. It will enter the nucleus.
