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Signalling via Enzyme-Linked Receptors

Learning Targets

  • How signalling via enzyme-linked receptors work, focusing on Receptor Tyrosine Kinases (RTKs).
    • How is the receptor activated?
    • How is RTK signalling organized?
    • RTK signalling induces widespread effects.
    • Example: signalling via insulin receptors.
  • Role of growth factor receptors and how they work.
  • MAP kinases cascade activation.
  • Role of GTP binding in signalling.
  • How growth factor signalling becomes 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

  • A patient, Mrs. Jones, has a lump on her breast and undergoes a biopsy.
  • The breast specialist suggests immediate treatment with Herceptin (Trastuzumab).
    • Why? How does this drug work?

Enzyme-Linked Receptors

  • Receptors are activated through ligand binding, such as growth factors and cytokines.
  • Ligand binding 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 Growth/Survival

  • 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, ligand binding causes receptor subunits to dimerize 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 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 uptake of glucose, amino acids & free fatty acids from blood.
  • Promotes synthesis & storage of carbohydrates, proteins, and lipids.
    • Binding of insulin to 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 multisite docking proteins for various effector molecules, enlarging 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.

Growth Factor Signalling and 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.
    • This ensures homeostasis of cell size, number, and function.

Epidermal Growth Factor Receptor (EGFR)

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

Major Pathways Activated by EGFR

  • 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:
    1. Protein phosphorylation.
    2. GTP binding.
  • Protein in an 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 is promoted by GEF = Guanine nucleotide exchange factors.
  • OFF SWITCH: Activation is 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.

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 initiation of sequential phosphorylation/activation steps of the MAPK cascade.

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.
    • This makes them a target of an expanding class of anticancer therapies.

Hyperactivation of EGFR Signalling in Cancer

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

EGFR Signalling: A Therapeutic Target

  • Anti-EGFR monoclonal antibodies inhibit ligand binding.
  • Small-molecule EGFR tyrosine-kinase inhibitors inhibit TK activity.
  • These block EGFR signalling to inhibit growth, tumour proliferation, and migration.

RTKs as Drug Targets

  • Examples of drugs that act against tyrosine kinases:
    • Small molecule drugs: Gleevec (imatinib), Iressa (gefitinib), Tarceva (erlotinib), Sutent (sunitinib), Sprycel (dasatinib), Tykerb (lapatinib).
    • Monoclonal antibody drugs: Herceptin (trastuzumab), Erbitux (cetuximab), Avastin (bevacizumab).

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 dimerization and thus inhibits activation of signalling.

RTK Inhibitors

  • Examples: Trastuzumab, Gefitinib, Erlotinib, Cetuximab, Bevacizumab.
  • Other inhibitors of the MAPK pathway:
    • B-Raf inhibitors: Dabrafenib, Vemurafenib.
    • Mek inhibitors: Refametinib, Selumetinib, Trametinib.
    • Raf inhibitors: Regorafenib, Sorafenib.

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.