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HUBS2206 Lecture 27 Notes: Signalling Via Enzyme-Linked Receptors (RTKs)

Signalling via Enzyme-Linked Receptors

Learning Targets

  • How signalling via enzyme-linked receptors work, focusing on RTKs.
    • How is the receptor activated?
    • How is RTK signalling organised?
    • RTK signalling induces widespread effects.
      • Example: signalling via insulin receptors.
  • What is the role of growth factor receptors? How do they work?
  • How exactly is the MAP kinases cascade activated?
  • What is the role of GTP binding in signalling?
  • How does growth factor signalling become deregulated in cancer?
  • Key concept: Receptor tyrosine kinase signalling plays an essential role in the regulation of cell growth and survival and is targeted for cancer treatment.

Case Study

  • Mrs. Jones has found a lump on her breast.
  • She undergoes a biopsy.
  • The breast specialist suggests that she immediately takes Herceptin (Trastuzumab).
    • Why? How does this drug work?

Enzyme-Linked Receptors

  • Receptors activated through ligand binding, e.g., growth factors, cytokines…
  • Binding of ligand induces conformational changes in the receptor.
  • Change in receptor structure activates an intrinsic enzyme activity associated with the receptor or promotes the binding and activation of an enzyme.
  • Enzymatic activation begins the signal transduction process.

Receptor Tyrosine Kinases (RTKs)

  • Major class of enzyme-linked cell surface receptors.
  • Have intrinsic tyrosine (Tyr) kinase activity.
    • Activated RTKs phosphorylate themselves on Tyr amino acids!
  • Contain:
    • An extracellular ligand-binding domain
    • One transmembrane domain per protein subunit
    • An intracellular protein tyrosine kinase domain
  • Family of about 60 human genes.

RTKs and Cell Signalling

  • RTKs typically transmit signals that promote cell growth and survival.
  • Activated by a large variety of secreted tissue cell-specific growth factors and hormones, like insulin.

Mechanisms of Activation of RTKs

  • For many receptor tyrosine kinases, binding of ligand to receptor causes the receptor subunits to dimerise and become activated.
  • Leads to autophosphorylation of Tyr residues within the kinase domain in the cytosolic tail of RTK.
  • Tyr phosphorylation outside the kinase domain creates high-affinity docking sites for the binding of a number of intracellular signalling proteins in the target cell.
  • Signalling proteins become activated – due to docking or Tyr phosphorylation.

Multiple Signals Generated by RTKs

  • RTK activation triggers the transient assembly of a large intracellular signalling complex.
  • This allows broadcasting multiple signals along multiple pathways to many destinations in the cell.
  • Distinct RTKs bind different combinations of signalling proteins, thereby inducing different responses.
  • Once cross-phosphorylated, the cytoplasmic tails of RTKs serve as docking platforms for various intracellular proteins involved in signal transduction.

Example: Insulin Signalling

  • After a meal, insulin stimulates the uptake of glucose, amino acids & free fatty acids from the blood.
  • Promotes synthesis & storage of carbohydrates, proteins, and lipids.
    • Binding of insulin to the receptor promotes Tyr autophosphorylation of the receptor.
    • Leads to Tyr phosphorylation of specific proteins called insulin receptor substrates (e.g., IRS-1).
    • Phosphorylated IRS serve as multi-site docking proteins for various effector molecules.
    • Allows enlargement of the size of the signalling complex.

Insulin's Effects

  • Insulin initiates a wide variety of growth and metabolic effects.
  • Activation of several signalling cascades leading to widespread effects (tissue-specific).
    • Rapid effects: e.g., glucose uptake, activation and inhibition of enzymes.
    • Long-lasting effects: e.g., protein synthesis (metabolic enzymes), cell growth.

Insulin Signalling

  • A complex pathway involving various proteins and molecules, resulting in diverse cellular effects such as glucose uptake, protein synthesis, and metabolic regulation.

Growth Factor Signalling and Significance in Cancer

  • Normal tissues carefully control the production and release of growth-promoting signals that instruct entry into and progression through the cell growth and division cycle → ensures homeostasis of cell size, number, and function.

Epidermal Growth Factor Receptor Activation

  • EGFR is a member of the HER family of 4 human epithelial receptors (EGFR or HER1, HER2, HER3, and HER4).
    • Each activated by specific ligands, except HER2 (no ligand required).
  • Dimerisation required for activation.
    • If the same receptor → homodimerization.
    • If a different receptor → heterodimerization.

Major Pathways Activated by EGFR Signalling

  • MAPK/ERK cascade
  • PI-3 kinase / Akt pathway
  • JAK/STAT pathway
  • Lead to enhanced growth and survival

Molecular Switches in Cell Signalling

  • Two main molecular switch mechanisms to turn on or off proteins:
    • Protein phosphorylation
    • GTP binding
      • Protein in the inactive state is bound to GDP.
      • Exchange of GDP for GTP leads to protein activation.
  • Two types of GTP-binding proteins:
    • Large heterotrimeric G proteins: transduce signals from G-protein coupled receptors.
    • Small monomeric GTPases: transduce signals from enzyme-linked receptors.

GTP Binding as a Signalling Switch

  • ON SWITCH:
    • GDP is exchanged for GTP, resulting in protein activation.
    • Exchange promoted by GEF = Guanine nucleotide exchange factors.
  • OFF SWITCH: Activation short-lived!
    • GTP binding proteins have intrinsic hydrolysing activity (GTPase activity).
    • GTP hydrolysis leads to inactivation, thereby terminating signalling.
    • GTPase-activating or GTPase-accelerating proteins called GAP increase the rate of GTP hydrolysis, accelerating inactivation.

The MAP Kinase Activation Pathway

  • RTK phosphorylation results in the recruitment of an adapter protein called Grb2 (growth factor receptor-bound protein 2).
  • Grb2 recruits SOS, a GEF that promotes the exchange of GDP to GTP on Ras (small GTPase) leading to Ras activation.
  • This leads to phosphorylation and activation of the kinase, Raf (or B-Raf), and the initiation of sequential phosphorylation/activation steps of the MAPK cascade.
    • You need to know the mechanisms of this cascade.

Abnormal EGFR Activation in Cancer

  • Abnormal, persistent activation of EGFR is intimately linked to cancer.

EGFR Signalling and Cancer

  • RTKs can be constitutively activated by mutations that induce malignant cell transformation.
  • Deregulation of signalling by EGFR or family members is a common feature in several human malignancies, including lung, breast, colorectal, prostate, and head and neck cancer.
    • Target of expanding class of anticancer therapies.

Hyperactivation of EGFR Signalling in Cancer

  • Overexpression of EGFR - often due to gene amplification.
  • Mutations in receptor and signalling proteins leading to constitutive activation.
  • Increased secretion of growth factors by tumour cells which can stimulate EGFR (autocrine).

EGFR Signalling: A Therapeutic Target

  • Anti-EGFR monoclonal antibodies and small-molecule EGFR tyrosine-kinase inhibitors block EGFR signalling to inhibit growth, tumour proliferation, and migration.

RTKs as Drug Targets

  • Cancer drugs that act against tyrosine kinases include small molecule drugs and monoclonal antibody drugs that target various RTKs.

Case Study: Herceptin

  • Biopsy has shown invasive cancer cells.
  • Genetic testing shows that the breast cancer is HER-2 (human epidermal growth factor receptor 2) positive.
    • ~1 in 5 women with breast cancer are HER2+.
  • Herceptin: Monoclonal antibody inhibits ligand-independent HER2 dimerisation and thus inhibits activation of signalling.

RTK Inhibitors

  • Various inhibitors target different components of the MAPK pathway.

Summary

  • RTK signalling is a key driver of cell signalling and is often hijacked in cancer.
  • Activated RTKs or downstream signalling proteins are targets for anti-cancer therapies.
  • RTKs activate downstream pathways via phosphorylation or GTP binding.
  • The MAPK pathway is an essential signalling cascade for cellular growth and survival, activated downstream of multiple RTKs, including EGFR/HER.