Receptor Tyrosine Kinases Flashcards
(38 cards)
What are kinases and phosphatases?
Kinases catalyses the transfer of a phosphate group
- Protein kinases (receptor tyrosine kinases) - 1000 different types
- Lipid kinases
Phosphatases catalyze the removal of a phosphate group. Phosphorylation is a major regulator (30% of mammalian proteins are phosphorylated)
Which amino acids can be phosphorylated?
- Serine and threonine (similar structure)
- Tyrosine
They all have a free hydroxyl group that can be phosphorylated.
What are the types of receptor tyrosine kinases ligands?
- EGF stimulate the proliferation of various cell types [EGF receptor]
- Insulin-like growth factor IGF1 and IGF2 stimulate cell growth [IGF receptor]
- Nerve growth factor stimulate neuron survival [TrKA]
- Insulin stimulates carbohydrate utilization and protein synthesis [Insulin receptors]
- Platelet-derived growth factor stimulates survival, growth and proliferation of various cell types [PDGF receptors]
- Macrophage-colony-stimulating factor stimulates macrophage proliferation [M-CSF receptor]
- Fibroblast growth factors provide inductive signals in development to stimulate/inhibit [FGF receptor]
- Vascular endothelial growth factor stimulates angiogenesis [VEGF receptor]
- Ephrins stimulate angiogenesis, guides cell and axon migration [Eph receptors A and B]
What is the biological importance of receptor tyrosine kinases?
1) Can function as oncogenes
2) FGF receptors important for wound healing
3) VEGF receptors important for angiogenesis
Inhibited in diabetics and cancer patients.
4) NGF receptors important for neural survival
How were FGF receptors found to be important in wound healing?
- In vitro: fibroblasts isolated and grown in culture. Petri dish filled is scratched in inflict insult. Cells move to fill the gap. After 24 hours the gap has been closed in control cells.
- Tissue genetically engineered to have a lack of FGF show a delay in wound healing. Leaving it long enough the wound will still heal
What are the common features of receptor tyrosine kinases?
20 families of RTKs (58 in total). All contain cysteine-rich domains, immunoglobulin-like and fibronectin-like. Similar pathways in cytosolic domain but have specificity in the extracellular domain.
-Extracellular binding site often has immunoglobulin or fibronectin domains and contains ligand-binding domain
-A single transmembrane domain
-Intracellular domain includes tyrosines that can be cross-phosphorylated (protein tyrosine kinase)
All TKDs have an N-loop, C-loop and an actvation loop. They are all cis-autoinhibited
How are receptor tyrosine kinases activated?
Monomeric receptor binds to ligand in ECD which leads to dimerisation/oligomerisation. Two receptors are close together so they tend to phosphorylate each other (crossphosphorylation). This allows the kinase domain to become constitutively active whereas before the ligand can flick between being active/inactive.
What are the types of ligand-induced dimerisation that can occur in RTKs?
- TrkA receptor NGF is a dimer ligand so it has 2 binding sites so requires 2 RTKs. Acts as an adaptor to bring the 2 ICDs together but the ECD don’t come into contact.
- In Kit, SCF is also a dimer. It binds to and induces a structural change in the ECD, contact between the two.
- FGFR requires HSPG/herarin for the binding of the ligand to the receptor. Part of ECDs comes together.
- EGFb dimer contains 2 single ligands and binds to ECD to induce dimerisation and a ‘sticky’ conformational change.
What is the downstream effect of RTK activation?
Creates phosphorylated tyrosine domains of the C terminal chain which can act as a binding site for downstream signalling transduction molecules. PLCy, Grb2, Shc all bind via SH2 domains. This can cause relocalisation to the plasma membrane so that further substrates can bind.
What is the insulin receptor like?
Heterotetramer (2a and 2B)
Insulin ligand binding to a subunit induces structural change which brings the receptor together, this activates the B subunit which phosphorylates Tyr residues on cytoplasmic domains and downstream substrates. Transphosphorylation disrupts the cis-autoinhibitory interactions. N-loop also reorientates enabling stablisation of the ATP binding site.
- Regulatory loop which contains a number of tyrosines
- N loop composed of mostly a-helices and b-sheets
- C loop is entirely a-helices
How does a kinase domain become active in the insulin receptor?
Kinase domain needs a cavity for the substrate and the ability to bind to ATP. Active when both of these are bound. In an inactive version the regulatory loop is present where the substrate or ATP would be, preventing activation.
Phosphorylation frees up the binding sites and allows the substrate to bind.
Same for kit (reg loop is present between kinase domain and ECD) and Tie2 (reg loop at C terminus).
How is EGFR activated?
The EGFR doesn’t have an regulatory loop that is phosphorylated. Instead, there is allerosteric activation. The tyrosine kinase domain forms an asymmetric dimer in which the c lobe of one TKD (activator) makes intimate contacts with the n lobe of the other TKD (receiver). This contact causes conformational change in the n lobe of the receiver kinase that disrupts cis-autoinhibitory interactions, allowing the activation loop to free up the substrate binding site.
How can SH2 domains bind to phosphorylated tyrosines?
Critical residues in the SH2 domains which gives specificity to phosphorylated tyrosines. Argenine and lysine interact with the aromatic ring of the phosphorylated tyrosines. Arg is positively charged, aromatic ring is negatively charged. This is called the cation-Pi interaction. Another postivitively charged Arg in the SH2 domain is in the right position to bind to the negative phosphate.
Removing Arg means phosphorylated tyrosine can’t bind.
How can EGF receptor activation lead to Ras activation?
EGF monomers dimerise and cross-phosphorylate. Grb2 binds to the SH2 domain of phosphotyrosine bringing it to the membrane. Grb2 binds to SOS via SH3 domain. SOS is the substrate for Ras, it promotes the dissociation of GDP from Ras. GDP removed, GTP binds and activates Ras. SOS dissociates.
How were drosophila used to elucidate the action of activated RTK causing activated Ras?
Drosophila ommatidia eye model. Composed of 8 different neurons (R1-8) plus 14 other cells. The RTK sevenless is decidated to the development of R7 = homolog for EGF receptor. In WT boss (bride of seveless) is the ligand for sevenless to induce the R& neuron. Phosphotyrosines bind to Grb2 via SH2 domains. SOS (son of sevenless) binds Grb2 and acts as an exchange factor to activate Ras.
Mutant SOS, Ras is not activated and R7 is not induced.
Rescue experiment: introduce constituatively active Ras to bypass sevenless signalling, R7 neuron introduced.
What is the neurotrophic hypothesis?
Targets of innervation secrete limiting amounts of survival factors to generate a balance between the size of the target organ and the number of innervating neurons.
Pheochromocytoma cell line (Pc12 cells) has EGF and NGF receptors. You can generate two different cell actions (proliferation and differentiation) but the downstream signalling is the same.
Likely that there’s a network of pathways leading to these different outcomes.
How can EGF and NGF cause different cell action through the length of the signals?
Both receptors undergo ligand induced endocytosis but the EGFR is transported to the lysosomes and degraded (shutting off the signalling). It is ubiquinated and will be sorted into intraluminal vesicles of multivesicular bodies via HRS and the ESCRT complexes. NGFR are predominantly recycled to the plasma membrane.
How do EGF and NGF differ in their effect on Ras/MAP kinase pathway?
Both receptors activate ERK (aka MAPK) via the kinase RAF. NGF activates ERK and PKC, PKC phosphorylates Raf Kinase inhibitory protein. This will dissociate from RAF which will then be phosphorylated by ERK [POSITIVE FEEDBACK LOOP].
EGF activates the expression of MAP kinase specific phosphatases called dual-specificity phosphatases. This will lead to dephosphorylation of ERK and will eliminate the positive ERK-RAF feedback loop. MAPK activity is transient [NEGATIVE FEEDBACK LOOP].
At low levels of ERK activation FOS is rapidly degraded. NGF stabilises FOS by ERK-mediated and Rsk2 mediated phosphorylation. MAPK activity is sustained. Sustained FOS activity resullts in induction of gene expression programmes that lead to differentiation and neurite outgrowth.
How are the numbers of receptor tyrosines controlled?
Ubiquintination targets receptors to the lysosomes, promoting their downregulation. CSF, EGFR, HGFR and PDGFR are all substrates for ubiquitination. Ubiquitination requires uniquitin-activating enzyme (E1), uniquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3). Monoubiquitination of EGFR and PDGFR is sufficient for their degradation. Other RTK that are not ubiquitinated, are sorted into multivesicular bodies to be recycled back to the membrane.
How can dysregulation of RTK cause disease?
Enhanced activity of RTKs through loss of negative regulation can cause disease. Deregulation of RTK can lead to ligand-independent activation or enhanced catalytic activity. RTK control cell proliferation, cell differentiation, cell migration and cell survival.
What is Cbl?
Family of ubiquitin-protein ligases (E3). Bind to the N terminal of the tyrosine kinase and transfer ubiquitin residues. C-cbl overexpression increases the rate of RTK internalisation at the membrane. In the case of Mer receptor, the tyrosine kinase binding domain of Cbl binds to a juxtamembrane tyrosine for ubiquitination and degradation.
How can changes in Cbl cause cancer?
-If the CSF-R1 without c-Cbl TKB domain is a viral oncogene
-APS (adaptor protein with PH and SH2 domains) is required to recruit c-Cbl to SCFR and PDGFR. When APS binding sites are missing in the cancer strain v-kit.
-The EGFR mutant lacking Cbl binding sites are oncogenic.
-Overexpression of HER2 causes EGFR/HER2 heterodimers to be formed causing EGFR to escape ubiquitination
-Chromosomal translocation - fusion of a protein dimerisation with the cytosolic kinase domain of the receptor causes receptor to be constitutively active.
[Trastumzumab is used with chemo to recruit Cbl to downregulate the overexpressed RTKs]
How can ligand-induced dimerisation structure vary between different receptors?
Two extremes: entirely ‘ligand-mediated’ and receptors make no contact or entirely ‘receptor-mediated’ and the ligand makes no contact. Or both.
How does FGF perform ligand-induced dimerisation?
FGF uses a combination of bivalent ligand binding, direct receptor-receptor contacts and the involvement of Heparin as an accessory molecule. Each FGF contacts both receptors molecules in the dimer forming a major interaction with one and a minor interaction with the other. The interactions stablilise the receptor. Acid-box binds to a postively charged ‘canyon’ within the receptor which would otherwise accomodate heparin autoinhibition.
