L18 - Receptor Tyrosine Kinases Flashcards
How many families of RTKs
16 families
Ligands can be of what two broad calss
Hi or lo affinity
What is the structure of the EC domain of the RTK
These vary greatly
What activity to the IC domain of the RTK have
Kinase activity
How many TMD of the RTK
What is the strcutre, what is the question this produces
1
Lacks structure
Question: How is the ligand binding transduced to the inside of the cell
RTK ligand has the abiliy to …
What can this do
DImerise and facillitates receptor dimerisation
May also reorient existing receptor oligomers
Describe what happens to the RTK once the ligand is bound
Kinase domains phosphorylate each other
Desribe the effects (3) once the kinase domains have phosphorylated each other
Increases the activity of the kinase
Stabilises the receptor in the active state
Casues the kinase to phosphorylate other tyrosines in the receptor to create a docking site
Tools for RTK analysis exploit …
Dimerisation
Describe how a dominatn geative RTK would be produced
Using genetic engineering to make DNA encoding a receptor which is MUTATED IN THE KINASE DOMAIN
Expressed at high levels and poisons the endogenous receptor
How is a constiuently active RTK produced
Use genetic engineering to make an RTK lacking the homodimerisation domain
DNA expressed at normal levels and is ligand independent
What are HSPGS
Multifunctional extracellular matrix components
Describe the types of protein core in HSPGs (3)
Transmembrane
Tethered
Secreted
What happens to the sugars on HSPGs
They can be moified
What is one of the most common modifcations on the HSPG sugar
Sulphation
What does modificaiton of the HSPG sugar result in
Results in a code that creates binding sites for specific proteins
What is a protein which commonly binds to HSPG
FGF2
Example of a secreted HSPG
Perlecan
Example of a tethered HSPG
Glypican
Example of a transmembrane HSPG
Syndecan
What occurs when FGF binds to its receptors
FGF and its receptor form a complex with HSPGs
FGF first forms oligomers of HSPGs
How does the activated receptor transduce the signal
Activated receptor recruits other proteins to the membrane
These protiens bind to phosphotyrosines
RTK have two binding sites - name them
Binding site for phosphotyrosine
Binding site for amino acid side chain
The SH2 domain in the RTK recongises which sequence
Phosphtyrosine - glutamic acid - glutamic acid - isoleucine
What 2 branches are there in the RTK pathway
RAS
PI3 kinase
What 3 proteins can bind to the PDGF receptor
PI3 kinase
GTPase activating protein (GAP)
Phospholipase C-gamma
PI3 kinase and PLC-gamma are part of which pathway
Inositol lipid pathway
What is the GTpase activating protein part of which pathway
RAS/MAP kinase pathway
What are some of the family members of the Ras family
H-RAS K-RAS N-RAS
Rheb
Rep1
Functions of H-Ras K-Ras and N-Ras
Relays signals from RTKs
Functions of Rheb
Activates mTOR to stmulate cell growth
function of Rep1
Activated by a cAMP dependent GEF
Influences cell adhesion by activating integrins
Family members of Rho
Rho Rac cdc42
functions of rho rac and cdc42
Relay signals from surface receptors to the cyotskeleton and elsewhere
What binds causing the coupling of the receptor to inactive ras
GRB2 and Sos(GEF)
What is the effect of GRB2 and Sos(GEF) bidning
Couples the receptor to inactive ras
What does Sos promote
What type of molecule is it
Promotes dissociation of GDP from Ras
GEF (gunaine nucelotide exhcnage factor)
What occurs after GDP has dissociated
GTP binds to Ras
Sos dissociates from the now active Ras
Describe the MAP kinase pathway which is downstream of the activated Ras
GTP-Ras activates MAP-KKK
MAP-KKK –> MAP-KK (phosohrylation consuming ATP)
MAP-KK –> MAP-K (phosohrylation consuming ATP)
MAP-K can then phosphorylate target proteins
What are the two targets for MAP-K what does phosphorylation at each cause
protein - changes protein activity
Gene regulatory proteins - changes in gene expression
MAP-K pathways is an example of a __________
What are the properites of this
Cascade
Rapid and transient response
What occurs at each stage of the MAP pathway
Amplificaiton
What methods may be used to study signalling
Visualisation or detection of interations
Chemical inhibitors
Misexpression or overexpression
Genetic methods
what types of visualisation methods may be used to study signalling
Biochemical methods (columns Fret (in vivo)
Descruve why grey scale camera are used for flouresecence microscopy
Colour cameras are not as sensitive
What do dichroic mirrors relfect
Shorter wavelengths
What is the light source used in flourescence microscopy
Laser or a mercury lamp
What are the exictation and emission wavelengths of Blue flourescent protein ECFP
Exitate at 410 nm
emmit at 480 nm
Descirbe the structure of a fluroescent fusion protein
Protein - peptide linker and flourophore
Blue flouresecnet protein
EXCITE COLOUR _______
EMITT COLOUR _______
violet
Blue
Green flouresecnet protein
EXCITE COLOUR _______
EMITT COLOUR _______
Blue
Green
In Fret if there is no interaction what will be seen why will this happen
Violet light excites the BFP (blue) causes emission of blue light
No GFP present so all observed is the blue light
In FRET if there is an interaction describe what will be seen, why does this happen
Violet light excites the BFP - emission of blue light which then excites the GFP causing emssion of green light
Green light is detect
What does FRET stand for
Forster resonance energy transfer
Describe how FRET may be used for protein and substrate
BFP - protein
GFP - substrate
If interaction between protein and sunstarate green light will be seen
Describe how FRET may be used for a ligand
BFP and GFP either side of the ligand binding site
If ligand binds these domains will be brought closer together
If ligand binds - FRET
Descibe how FRET may be used for a sensory domain
BFP - sensory doamin - substrate - GFP
If substrate binds to sensory domain then BFP and GFP brought closer together and FRET
Describe how FRET may be used with a proteease
GFP and BFP connected with a peptide linker
If cleavage occurs there will be no FRET
FRET will only occur if there is a link between the BFP and the GFP
What type of afifnity do HSPGs have for RTKs
Low affinity
