lecture 6- Receptor Tyrosine Kinases

Q: What are the key features of kinase-linked receptors?

A: Large proteins, up to 1000 residues, with one membrane-spanning helix. They mediate growth factors, cytokines and hormones and regulate division, growth, differentiation, inflammation, repair, apoptosis and immunity.

Q: What are the three types of kinase-linked receptors? And what do they do?

A: RTKs (receptor tyrosine kinases), serine/threonine kinases and cytokine receptors. RTKs - Receptors with an intracellular tyrosine kinase domain that phosphorylates tyrosine residues. Examples: EGF, NGF and insulin receptors. Second type - Similar to RTKs but phosphorylate serine/threonine residues. Main example: transforming growth factor (TGF) receptor. Cytokine receptors - Receptors with no intrinsic enzyme activity. They activate cytosolic tyrosine kinases such as JAK. Examples: interferons and colony-stimulating factors.

Q: What are RTKs usually associated with?

A: Long-term changes in cell function where gene expression is required, e.g. proliferation and differentiation. Examples: EGF—proliferation; VEGF—angiogenesis; insulin—glucose homeostasis.

Q: What is the basic structure of an RTK?

A: An extracellular ligand-binding domain, a single hydrophobic transmembrane α-helix, and an intracellular tyrosine kinase enzyme intrinsic to the receptor. (Come back to diagram on slide 9)

Q: What is the first step in RTK activation?

A: The receptor has low or no kinase activity in an unphosphorylated/blocking conformation. Ligand binding begins the activation process. (Come back to diagram on slide 10)

Q: What happens when a ligand binds an RTK?

A: Ligand binding promotes receptor dimerisation, bringing two receptor chains together. (Come back to diagram on slide 11)

Q: What does RTK dimerisation cause?

A: Receptor dimerisation causes a conformational change, resulting in transphosphorylation of intracellular tyrosine residues. (Come back to diagram on slide 12)

Q: What is the role of phosphorylated tyrosines?

A: Phosphorylated tyrosines function as docking sites for intracellular proteins which relay and amplify the downstream signal. (Come back to diagram on slide 13)

Q: What are the main HER receptor features shown?

A: HER1 binds EGF, HB-EGF and TGFα; HER2 is always active and dimerises with HER1, HER3 and HER4; HER3 has weak kinase function and is active when complexed with HER2; HER4 binds HB-EGF and neuregulins. (Come back to diagram on slide 14)

Q: How does PDGF activate its receptor?

A: PDGF exists as a preformed dimer with two receptor-binding sites, so the agonist directly cross-links two PDGF receptor chains. (Come back to diagram on slide 15)

Q: What is required for efficient FGF receptor activation?

A: FGF binds to the extracellular domain of the receptor and to heparan sulfate, which is essential for efficient activation. (Come back to diagram on slide 16)

Q: What very important hormone acts at an RTK?

A: Insulin acts at a receptor tyrosine kinase.

Q: What is the key point from the insulin discovery section?

A: Experiments showed that an extract from the islets of Langerhans could keep diabetic dogs alive; purified extract subsequently saved people dying from diabetes. (Come back to diagram/image on slide 18)

Q: How do insulin and IGF-I activate their receptors?

A: Insulin/IGF-I are soluble monomeric polypeptides. Their receptors are disulphide bond-linked preformed heteromers; binding to α subunits causes a conformational change transmitted to β subunits, which transphosphorylate each other. (Come back to diagram on slide 19)

Q: What are the major pathways downstream of RTKs?

A: RTKs signal through docking/adaptor proteins, small GTPases, MAPKs, PI3K and phospholipids, ultimately producing gene transcription and biological effects. (Come back to pathway diagram on slide 20)

Q: How do phosphorylated tyrosines recruit intracellular proteins?

A: Phosphorylated tyrosines act as high-affinity docking sites. SH2 domain proteins contain a 100-AA recognition site and bind selectively, making the response to receptor activation specific. (Come back to diagram on slide 22)

Q: What happens after SH2 domain proteins bind RTKs?

A: Many SH2 proteins are enzymes such as kinases or phospholipases. They can activate or inhibit transcription factors, which migrate to the nucleus and alter gene transcription. NFκB is an example. (Come back to diagram on slide 23)

Q: What state is inactive Ras in?

A: Inactive Ras has GDP bound and is therefore in the OFF state at the plasma membrane. (Come back to diagram on slide 25)

Q: How does Grb2 interact with an activated RTK?

A: Phosphorylated tyrosines act as docking sites for the SH2 domain on the adaptor protein Grb2. (Come back to diagram on slide 26)

Q: What does Grb2 recruit during Ras activation?

A: Grb2 interacts with a Ras guanine nucleotide exchange factor called Sos. (Come back to diagram on slide 27)

Q: How does Sos activate Ras?

A: Membrane-localised Sos exchanges GDP for GTP, removing GDP to allow GTP binding. Ras becomes active when GTP is bound. (Come back to diagram on slide 28)

Q: How does Ras activate the MAPK pathway?

A: GTP-bound Ras recruits Raf to the plasma membrane. Raf activates MEK; MEK activates MAP kinase; MAP kinase alters multiple enzymes and transcription factors to stimulate cell proliferation. (Come back to pathway diagram on slide 29)

Q: What other signalling pathway can RTKs activate?

A: RTKs also signal through phosphorylated phospholipids derived from phosphatidyl inositol. PI3K is positioned near substrates on the cytosolic face of the plasma membrane and produces PIP3. (Come back to diagram on slide 30)

Q: How are RTKs linked to PI3K activation?

A: Phosphorylated receptor and GTP-Ras recruit PI3-kinase to the membrane, positioning it near substrates on the cytosolic face of the plasma membrane. (Come back to diagram on slide 31)

Q: How does PI3K activate PKB/Akt?

A: PI3K phosphorylates membrane lipid PIP2 to PIP3. PIP3 acts as a second messenger and activates PKB/Akt. PDK1 phosphorylates a Thr residue and PDK2 phosphorylates a Ser residue of PKB. (Come back to pathway diagram on slide 32)

Q: What are the effects of PKB/Akt activation?

A: PKB/Akt phosphorylates and alters the activity of multiple enzymes and transcription factors to stimulate cell survival, cell growth and migration. Targets shown include mTOR, eNOS and transcription factors. (Come back to diagram on slide 33)

Q: Why can one ligand produce different effects through an RTK?

A: Different phosphorylated tyrosine residues bind specific SH2 domain-containing proteins, so one ligand can have different effects depending on the SH2 protein bound to the receptor.

Q: How is HER2 involved in cancer?

A: HER2 is overexpressed in 20–30% of breast cancers and is constitutively active, causing inappropriate amplification of mitogenic signalling, aggressive tumour growth and increased risk of metastasis. (Come back to diagram on slide 35)

Q: Which drugs target HER2/HER receptors?

A: Herceptin/Trastuzumab is a monoclonal antibody that binds and inactivates HER2. Lapatinib is a HER-targeted tyrosine kinase inhibitor molecule.

Q: How can HER1 mutations cause unrestricted signalling?

A: Some tumour HER1 mutations delete much of the EGF-binding domain, making the receptor incapable of binding EGF. It can then constitutively dimerise independently of ligand binding, causing unrestricted trans-phosphorylation and downstream signalling. (Come back to diagram on slide 36)

Q: How is VEGF signalling linked to cancer?

A: The lecture identifies VEGF signalling as a pathway involved in cancer and shows VEGF inhibitors as a therapeutic approach. (Come back to diagram on slide 37)

Q: What does the lecture highlight about TKIs?

A: Tyrosine kinase inhibitors (TKIs) can have side effects. (Come back to diagram on slide 38)

Q: How can RTK signalling contribute to hypertension?

A: RTK-associated signalling can shift normal vascular homeostasis towards vascular remodelling, increased stiffness and vascular dysfunction. The pathway shown includes PKB/Akt, mTOR, eNOS and transcription factors. (Come back to diagram on slide 39)

Q: What therapeutic target is shown for RTK-related signalling?

A: mTOR is shown as a drug target in the RTK signalling pathway. (Come back to diagram on slide 40)

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