Lecture 18: RTKs Flashcards
How many families of RTKs are there?
16
What are the functions of EGF?
promote:
- cell growth
- cell survival
- proliferation
What is the structure of RTKs?
- extracellular domain varies depending on the receptor e.g. cysteine rich domains, Ig-like domains, repeat sequences = ligand binding sites
- intracellular domain has kinase activity
- single transmembrane domain
What is the purpose of ligand dimerisation during canonical RTK activation?
- facilitates receptor dimerisation/oligomerisation - receptors in close proximity so the receptors can interact with each other
- reorientates receptor oligomers so their active domains are face to face
- increased kinase activity
- autophosphorylation of tyrosines creating docking sites
- kinase domains phosphorylate each other. stabilises receptor in active state.
Creating dominant negative RTK
- normal receptor with mutated tyrosine kinase domain overexpressed
- blocks activity of functional RTK during dimerisation - won’t phosphorylate to activate it
Creating constitutively active RTK
- receptor with homodimerisation domain instead of ligand binding domain expressed at normal levels
- ligand independent
- cells rapidly divide
HSPG with transmembrane protein core
Syndecan
HSPG with tethered protein core
Glypican
Secreted HSPG
Perlecan
Structure of HSPGs
long chain of sugars on a protein core. the sugars are modified by sulphation, resulting in specific binding sites for specific proteins (confers specificity)
Function of HSPGs
co-receptor facilitating e.g. FGF ligand dimerisation
What phosphopeptide does the Src SH2 domain recognise?
phosphotyrosine- glutamic acid - glutamic acid - isoleucine
Which intracellular signalling protein in the RTK pathway activates RAS/MAP kinase pathway?
GAPs e.g. GRB2
Which intracellular signalling protein in the RTK pathway activates inositol lipid pathway?
PI3K and PLC-gamma
How does the Ras signalling cascade become activated?
- inactive Ras is tethered to the membrane
- GRB2 binds to docking sites on receptor
- Sos binds to GRB2, coupling inactive Ras to the receptor
- Sos promotes dissociation of GDP from Ras
- GTP binds to Ras - Ras activated
- Sos dissociates from inactive Ras
- Ras initiates signalling cascade
Mutation analysis
Cloning sequence mutations to identify signalling components
Fluorescence microscopy camera
- strong light source = laser or mercury lamp is used
1. light passes through excitation filter
2. light hits a mirror reflecting down on the specimen
3. light hits a dichroic mirror which reflects shorter wavelengths and allows longer wavelengths to pass through
5. emitted light passes through barrier filter and reaches the eyepiece - image = black and white
Fluorescent protein fusions
Protein bound to fluorophore via protein linker
What is the excitation wavelength for ECFP
410nm
What is the emission wavelength for ECFP
480nm
What is the purpose of FRET
- monitors protein-protein interactions in live cells
-
Describe how FRET occurs
- protein x bound to BFP excited by violet light and emits blue light
- protein y bound to GFP excited by blue light and emits green light
interaction? - protein x excited by violet light and emits green light
emission from protein x exits protein y due to FRET
How could FRET be used to study signalling?
- study conformational changes. ligand binding bringing 2 proteins together results in FRET
- study protein cleavage - protease cleaving interaction between 2 proteins results in no FRET occurring
