Receptor tyrosine kinases

What is the structure of receptor tyrosine kinases (RTKs)?

• More than 50 RTKs have been identified. Each has an extracellular region (binds ligand), a transmembrane region, and an intracellular tyrosine kinase domain.

• Types differ in their variety of extracellular regions, but the tyrosine kinase domains are fairly conserved.

How are RTKs activated?

• Ligand binding induces dimerisation of the RTK (ligands are often dimers).

• Autophosphorylation: each receptor trans-phosphorylates its partner on Tyr.

• Recruitment of phospho-Tyr binding proteins, and increased Tyr kinase activity towards exogenous substrates.

What are Src homology 2 (SH2) domains?

• SH2 domains are phosphotyrosine (pTyr) binding domains.

• It is 100 amino acid long, with a core which has a highly conserved consensus sequence Phe-Leu-Val/Ile-Arg (FLVR) sequence.

• It binds to pTyr-x-x-hydrophobic residue (X is any, but often hydrophobic).

• It consists of 2 alpha-helices surrounding a central beta-sheet. The peptide containing pTyr lies at right angles to the beta-sheet. 

How is SH2 specificity determined?

• SH2 binding specificity is determined by residues downstream of (C-terminal to) the pTyr.

• SH2 has 2 or more binding pockets which need to be filled simultaneously.

–    It has a universal pocket which binds pTyr.

–    It has specificity pockets which binds the residues downstream of pTyr.

• Different SH2-containing proteins (like GRB2, Src, or PI3K) have different shapes in their specificity pocket; they require a specific sequence of amino acids following the pTyr to fit.

• They preferentially bind Leu at +3 of pTyr.

What are Src homology 3 (SH3) domains?

• SH3 domains consist of a 60 amino acid sequence (found in Src, PLCgamma and GRB2).

• It binds to proline rich sequences, typically Pro-x-x-Pro (x = any but often hydrophobic or another Pro).

• It consists of 5 anti-parallel beta-strands forming 2 beta-sheets at right angles to each other. Each Pro binds in a hydrophobic pocket.

What is the platelet-derived growth factor receptor and what does it signal?

• Platelet-derived growth factor (PDGF) can signal to cause cell growth, cell survival or cell migration.

• It signals via RAS-MAPK (growth), PI3K-Akt (survival/growth) and PLCγ-PKC (migration/growth).

• Its extracellular region has many Ig-like domains.

• It plays a key role in mesenchymal cells and neurons. PDGF is usually produced by epithelial cells.

How does PDGFR signal via RAS-MAPK?

Pathway: PDGFR pTyr → GRB2 → SOS (GEF) → RAS (GDP → GTP) → RAF (MAPKKK) → MEK (MAPKK) → ERK (MAPK) → cytoplasmic and TF targets → cell growth

• GRB2 (adaptor) binds phospho-Tyr via its SH2 domain. Its 2 SH3 domains bind proline-rich residues in the GEF SOS.

• SOS exchanges GDP for GTP on inactive RAS → RAS is active.

• RAS binds the Ras-binding domain (RBD) of RAF. This pulls RAF out of the cytoplasm and locks it onto the inner side of the cell membrane. At the membrane RAF dimerises and autophosphorylates to become active.

• RAF phosphorylates MEK, which phosphorylates ERK.

• ERK has cytoplasmic and TF targets → triggers cell growth.

How is RAS regulated?

• Controlled by opposing actions of GEFs (mediate GDP/GTP exchange) and GAPs (promote RAS's intrinsic GTP hydrolysis, removing the terminal phosphate of GTP).

• RAS is mutated in ~30% of cancers into its GTP-bound form, constitutively activating signalling pathways.

How does PDGFR signal via PI3K-Akt?

• PI3K binds pTyr. It is made of p85 (regulatory; 2 SH2 domains) and p110 (catalytic).

• PI3K phosphorylates PIP₂ → PIP₃ in the membrane.

• PIP₃ acts as a temporary docking site for PDK1, Akt and mTORC2, which bind via PH domains.

• PDK1 and mTORC2 phosphorylate Akt at different sites. This dual phosphorylation is required for Akt signalling.

• Active Akt phosphorylates several targets.

–    BAD (pro-apoptotic) - phosphorylation sequesters BAD in a complex with 14-3-3. BAD was attached to the anti-apoptotic protein BCL-XL, so this sequestration releases BCL-XL

–    FOXO (apoptosis TF) - phosphorylation sequesters FOXO in the cytoplasm with 14-3-3, preventing transcription of pro-apoptotic genes.

How are PIP₃ levels regulated?

PIP₃ levels are regulated by the opposing activities of PI3K and PTEN (phosphatase and tensin homologue), which dephosphorylates PIP₃ → PIP₂.

How does RAS-MAPK signalling reinforce PI3K-Akt signalling?

RAS reinforces PI3K: the catalytic p110 subunit can bind activated RAS. This stabilises PI3K at the membrane, so its activity is maintained for longer.

How does PDGFR signal via PLCγ-PKC?

• PLCγ binds pTyr via SH2 and PIP₃ via PH.

• PLCγ cleaves PIP₂ → IP₃ + DAG.

• IP₃ travels to the ER and binds IP₃-gated Ca²⁺ channels → cytosolic Ca²⁺ rises.

• Ca²⁺ binds and activates PKC, which binds DAG in the membrane → fully active.

• PKC phosphorylates targets that promote cell growth and migration.

How is PKC primed before activation?

• Newly synthesised PKC is phosphorylated by mTORC2 at a hydrophobic motif, which stabilises its folding.

• It is then phosphorylated by PDK1 on its activation loop.

• This changes its shape and allows PKC to autophosphorylate itself = primed.

• mTORC2 and PDK1 are attached to PIP₃, so this pathway is linked heavily to PI3K.

What are phosphatidylinositols (PI) and how are they modified?

• Phosphatidylinositols = inositol head group + glycerol linker + fatty acid chains.

• PIP₂ is phosphorylated at positions 4 and 5. PI3K adds a phosphate at position 3 (→ PIP₃); this is reversible via PTEN.

• PLCγ cleaves the glycerol linker, giving diacylglycerol (DAG) and inositol trisphosphate (IP₃).

What is the insulin receptor?

• Another RTK, but with a distinct tetramer structure: 2 Tyr kinase domains that pass through the membrane and 2 extracellular domains, all joined by disulphide bonds.

• It is already dimerised, so does not undergo ligand-induced dimerisation.

How does the insulin receptor signal?

• Insulin binds the α subunit → autophosphorylation.

• The activated receptor recruits and phosphorylates the adaptor insulin receptor substrate (IRS).

• IRS binds the pTyr via a PTB domain (instead of SH2) and is anchored to the membrane via a PH domain binding PIP₂.

• IRS recruits PI3K → protein synthesis, glycogen synthesis, glucose transport. It also recruits SHP2 and GRB2-SOS (via SH2) → mitogenesis.

What it the PTB?

• Phosphotyrosine-binding domains (PTB) binds to Asn-Pro-x-pTyr.

• Its specificity determinants are upstream/N-terminal to pTyr.

• It consists of a beta-sandwich structure with 2 orthogonal beta-sheets and a C-terminal alpha-helix capping one end. pTyr fits in the cleft between the alpha helix and beta sheets.

How does the insulin receptor trigger glycogen synthesis and glucose uptake?

Glycogen synthesis: IRS → PI3K → PIP₃ → (PDK1 + mTORC2) → Akt ⊣ GSK3 ⊣ glycogen synthase (GS) → glycogen synthesis. Double negative = positive.

• GSK3 (glycogen synthase kinase 3) is itself an inhibitor of GS. Akt inhibits GSK3, so GS is released.

• Glucose uptake: Akt phosphorylates AS160, which normally binds GLUT4 vesicles to keep them inactive. Phosphorylated AS160 binds 14-3-3 (sequestration), releasing GLUT4 to translocate to the membrane. GLUT4 lets glucose enter cells.

How does insulin resistance arise?

• In type 2 diabetes, signalling from IRS is impaired.

• TNFR1 signalling can induce insulin resistance: it activates JNK, which phosphorylates Ser307 in IRS. This causes a conformational change that blocks IRS's PTB domain from binding the insulin receptor.

What is receptor-mediated endocytosis and what is its role?

Receptors can be removed from the cell surface by receptor-mediated endocytosis. It has 2 main functions:

–    Attenuates signalling by an activated surface receptor.

–    Allows receptors to interact with downstream signalling molecules.

• Mechanism: clathrin coats the inner leaflet beneath the receptors, contracts into a pit, then a vesicle that engulfs the receptors.

How is epidermal growth factor (EGF) receptor endocytosed?

• GRB2 is recruited to the pTyr on EGFR, and recruits CBL (ubiquitin ligase) via SH3 domains.

• CBL is phosphorylated on Tyr by EGFR, which lets it bind the CIN85-endophilin complex. This complex binds the membrane and promotes invagination.

• CBL mono-ubiquitinates the receptor, which recruits the adaptors EPS15 and epsin. They contract the clathrin → internalisation.

What are the fates of endocytosed receptors?

• Vesicles fuse with early endosomes. The acidic lumen disrupts the ligand-receptor interaction.

• Degradation: the late endosome delivers ligand and receptor to the lysosome, where lysosomal proteases degrade them.

• Recycling: the receptor can be returned to the membrane before delivery (between early and intermediate endosome states).

How does receptor endocytosis allow receptors to interact with downstream molecules?

Receptors can keep signalling from endosomes, activating the same pathways. Endocytosis is essential for sufficient signalling because it provides another branch of activation. Example: inhibiting PDGF endocytosis decreases MAPK signalling.

What determines the fate of the receptors?

• There is a balance between the two, and the ratio differs between receptors. For EGFR:

–    unbound receptors: ~75% recycled

–    EGF-bound receptors: ~75% degraded

• HER2/EGFR heterodimers are preferentially recycled compared with EGFR homodimers. The low pH in the endosome is more likely to dissociate heterodimers but not homodimers, so homodimers are more likely to be degraded.

• Increased HER2 expression → more HER2/EGFR recycling and less receptor degradation → increased cell growth (HER2 is a more potent growth stimulator than EGFR).

How is HER2 implicated in breast cancer?

• HER2 receptor numbers can increase >100-fold. HER2-positive cancers are ~30% of breast cancers.

• Overexpressed HER2 forms heterodimers with EGFR, or homodimers.

• No known growth factor ligand for HER2, so homodimers can signal independently of growth factor.

• Herceptin is a monoclonal antibody that blocks HER2 homodimerisation.