Receptor Tyrosine Kinases

Overview of Enzyme-Coupled Receptors:

RTKs are a major class of enzyme-coupled receptors, a group of cell-surface receptors that possess intrinsic enzymatic activity or are directly associated with enzymes.

• Like GPCRs (G-protein coupled receptors), enzyme-coupled receptors:

  • Are transmembrane proteins.

  • Bind extracellular ligands (e.g. hormones, growth factors).

  • Transduce signals across the plasma membrane to trigger intracellular responses.

• Key difference from GPCRs:

  • GPCRs have seven transmembrane α-helices and are activated by large conformational changes.

  • RTKs usually have one transmembrane domain and rely on ligand-induced dimerisation, not conformational shifts, for activation.

Structural Features of RTKs:

There are approximately 60 RTKs in humans, grouped into ~20 subfamilies (based on structure and ligand specificity).

• Extracellular domain:

  • Large and variable; responsible for ligand recognition.

  • Common motifs: immunoglobulin-like domains, cysteine-rich regions, and fibronectin type III-like domains.

• Transmembrane domain: single α-helix spanning the membrane.

• Intracellular domain:

  • Contains an intrinsic tyrosine kinase domain.

  • Capable of autophosphorylating specific tyrosine residues upon activation.

Activation Mechanisms:

General Activation Process:

• Ligand binding to extracellular domain induces receptor activation.

  • Activation stimulates the cytosolic kinase domain.

    • Tyrosine residues within the receptor’s intracellular region are phosphorylated.

      • These phosphotyrosine residues act as docking sites for intracellular signalling proteins.

Mechanism 1: Dimerisation and Trans-Autophosphorylation:

• Common in receptors such as the insulin receptor.

  • Steps:

    • Ligand binding brings two receptor monomers together → dimerisation.

    • Each kinase domain phosphorylates tyrosine residues on the other monomer (trans-autophosphorylation).

    • Phosphorylated tyrosines act as recruitment sites for downstream signalling proteins.

Mechanism 2: Conformational Activation (Asymmetric Dimerisation):

Seen in the EGF receptor.

• Two monomers dimerise upon ligand binding, forming an asymmetric dimer:

  • One subunit acts as the activator.

  • The other as the receiver, which undergoes a mild conformational change leading to activation.

The activated kinase domain then phosphorylates both itself and the partner subunit.

Specificity of Phosphotyrosine Binding:

• Signalling proteins recognise phosphotyrosine sites via surrounding amino acid context, ensuring specificity.

  • Each phosphotyrosine recruits specific proteins, giving rise to distinct signalling complexes and cellular outcomes.

Downstream Signalling Proteins and Complex Formation:

Activation of Signalling Proteins:

• Phosphorylation-dependent activation: this mechanism involves the direct phosphorylation of intracellular signalling proteins by the activated RTK.

  • Upon activation, the RTK’s intrinsic tyrosine kinase domain phosphorylates specific tyrosine residues on target proteins, such as IRS (Insulin Receptor Substrate) in insulin signalling.

    • This phosphorylation can either directly activate the enzymatic activity of the target protein or create novel binding sites for other downstream signalling molecules, thereby propagating the signal.

• Binding-dependent activation: the activation of signalling proteins occurs by their binding to the phosphotyrosine residues on the activated RTK or associated scaffold proteins, without requiring direct phosphorylation of the bound protein itself.

  • Such binding can induce conformational changes within the bound protein, leading to its activation or exposure of its active sites.

    • Alternatively, binding events can bring inactive proteins into close proximity, facilitating their interaction with other complex members and leading to their activation, often through allosteric regulation or induced fit mechanisms.

Scaffolding and Adapter Proteins:

• Scaffold proteins (e.g. IRS) provide additional phosphotyrosine sites, enlarging the signalling complex.

  • Adapter proteins (e.g. GRB2) bridge interactions:

    • SH2 domains bind phosphotyrosine residues.

    • SH3 domains bind proline-rich motifs in other proteins.

      • This modularity allows the formation of large multiprotein complexes for signal amplification.

Binding Domains:

• SH2 domain (Src Homology 2):

  • Binds specific phosphotyrosine motifs.

  • Has pockets for both the phosphotyrosine and adjacent side chains → high binding specificity.

• PTB domain (Phosphotyrosine-Binding):

  • Alternative motif for phosphotyrosine interaction.

  • Found in proteins such as IRS.

RTKs and Ras Signalling:

The Ras Superfamily:

• Small monomeric GTPases that act as molecular switches.

  • Active when bound to GTP, inactive when bound to GDP.

    • Families within the superfamily:

      • Ras family (H-Ras, K-Ras, N-Ras).

      • Rho family (Rho, Rac, Cdc42).

      • Others: Rab, Arf, Ran (not RTK-regulated).

Activation of Ras by RTKs:

• Mechanisms of activation:

  • Activate Ras-GEFs (promote GDP→GTP exchange → ON).

  • Inhibit Ras-GAPs (prevent GTP hydrolysis → remain ON).

• Predominantly occurs through Ras-GEF activation.

  • Steps:

    • Ligand activates RTK → autophosphorylation.

    • GRB2 binds RTK via its SH2 domain.

    • GRB2’s SH3 domains recruit SOS (Ras-GEF).

    • SOS catalyses GDP→GTP exchange on Ras.

    • Active Ras triggers downstream signalling cascades.

Evidence for Transient Ras Activation:

• FRET experiment demonstrated Ras activation kinetics:

  • Cells expressing Ras-YFP and GTP–red dye construct.

  • EGF addition (activates EGF receptor → RTK).

  • Ras activity rises rapidly (peak ~3 min), then falls by 6 min.

• Demonstrates transient and tightly regulated Ras activation following RTK stimulation.

Ras-Mediated Signalling Pathways:

MAP Kinase Pathway (RAF–MEK–ERK):

• Sequential activation cascade:

  • Ras activates RAF (MAPKKK).

    • RAF phosphorylates MEK (MAPKK).

      • MEK phosphorylates ERK (MAPK).

• ERK phosphorylates nuclear and cytosolic targets → regulates gene expression, cell proliferation, and survival.
  

Other Ras Effector Pathways:

• PI3K–AKT pathway: promotes survival and growth.

• Ral-GEFs: influence cytoskeletal rearrangement.

• PLC pathway: generates IP₃ and DAG, increasing Ca²⁺ and activating PKC.

• Ras therefore acts as a hub for multiple signalling outputs.

Ras in Cancer:

• Ras is mutated in ~30% of all human cancers.

  • Oncogenic mutations lock Ras in the GTP-bound (active) state → continuous proliferation signalling.

• Consequences:

  • Uncontrolled activation of MAPK and PI3K pathways.

  • Persistent cell division and survival signals → tumour formation.

• Other mechanisms of deregulation:

  • Overexpression/mutation of RTKs (e.g. EGF receptor, ErbB2/HER2).

  • Overproduction of growth factors.

• Therapeutic strategies:

  • Monoclonal antibodies (e.g. Herceptin) block receptor activation.

  • Tyrosine kinase inhibitors (TKIs) block catalytic kinase activity.

RTKs and PI3 Kinase Signalling:

Overview:

• PI3K (Phosphoinositide 3-Kinase) is activated by RTKs (and also GPCRs and Ras).

  • Unlike protein kinases, PI3K phosphorylates membrane phospholipids.

Mechanism:

• Converts PIP₂ → PIP₃ at the plasma membrane.

  • PIP₃ acts as a membrane-bound second messenger.

    • PTEN phosphatase converts PIP₃ back to PIP₂, terminating the signal.

Functional Significance of PIP₃:

• PIP₃ recruits proteins with Pleckstrin Homology (PH) domains (~200 human proteins).

• Key targets:

  • AKT (Protein Kinase B): promotes cell growth, metabolism, and survival.

  • SOS (Ras-GEF): links PI3K and Ras pathways.

• Persistence of PIP₃ ensures sustained signalling until dephosphorylated.

Crosstalk Between GPCR and RTK Signalling:

• Both can activate overlapping pathways:

  • Phospholipase C (β via GPCRs; γ via RTKs)

  • PI3K activation

• These pathways converge at shared effectors, illustrating extensive signalling network integration.

• Cellular outcomes depend on signal context, intensity, and duration.