Keywords

EGFR, Dimerization, Loose Linkage, Transmembrane Signaling, Receptor Tyrosine Kinase

Reference

DOI: 10.1128/MCB.00742-10

Abstract

The study reveals that in EGFR, ligand-induced dimerization does not require strict transmembrane domain alignment, challenging prior models of receptor activation. Linkage between EGF binding and tyrosine kinase activation is far looser than previously thought.

Pre-knowledge

  • Recombinant EGFR fragments with both extracellular (ECD) and TM domains dimerize better than ECD alone — highlighting TM domain’s importance.
  • All ErbB family TM domains can self-associate in bacterial membranes.
  • Distinct GxxxG motifs in ErbB TM domains mediate dimerization in bacteria — but unclear if essential for signaling in cells.

Cool Findings

EGF-bound dimeric EGFR ECD crystal structure (includes domain IV):

  • Heart-shaped dimer: EGF bridges domains I and III; domain II as major dimer interface.
  • Domain IV (rod-like) curves inward; small domain IV interface but brings Thr614 close (~8Å), possibly orienting TM domains.
  • 7-residue linker (615-621) connects domain IV to TM domain (TM starts at Ile-622).
  • Crystal structure finalized from residues 1-614; 615-619 disordered.

Disulfide Cross-linking of EGFR on Cells

  • Disulfide cross-linking shows EGF-induced dimerization aligns linker residues, but linker remains flexible.
  • TM domain cross-linking shows proximity at Ala-623, Thr-624, Val-627 — only first two helical turns, unlike strong 5-turn dimerization in glycophorin A.
  • Thus, EGFR TM domains only moderately associate upon EGF binding.


Figure : two models they proposed (Click to enlarge)

Experiments and Techniques

  • Coot for model building, PHENIX for refinement.
  • Disulfide cross-linking on cell surface using cysteine mutants (C3M and C6M).
  • Ba/F3 cells lacking endogenous ErbB ensure specificity of cross-linking data.
  • Extracellular vs. TM cross-linking adjusted based on cysteine accessibility.

Disulfide-Rosetta Models of TM Domain Interfaces

  • Two dimer models produced via Rosetta — both supported by disulfide cross-linking patterns.
  • αG helix and Tyr/Phe residues contribute to transient interface.
  • BUT — even with specific models, functional data suggest no unique interface is required.

Leucine Scanning of TM Domain

  • Mutating potential TM interface residues (GxxxG-like motifs) to leucine — “Leucine scanning.”
  • No loss of autophosphorylation; some mutants even enhanced signaling.
  • Thus, EGFR does not require specific TM interfaces for kinase activation — major revision of previous models.

Fancy Thought / Conclusion

“The primary function of domain IV may be to provide orientation between domain III and the TM domain to bring the two TM domains of a dimer into close proximity.”

And yet: “We found no evidence of a requirement for a specific domain IV interface in EGF-stimulated kinase activation.”

Some Other Thoughts to Revisit

  • Their method of quantifying EGFR per cell — interesting and familiar. Worth a revisit when brain is fresh (not coffee-fueled).
  • Disulfide cross-linking combined with Rosetta modeling — good approach for structural-functional linkage.
  • Conceptually: EGFR activation is more about “approximation and stochastic interactions” than rigid alignment.
  • Could challenge classic textbook diagrams of “perfectly aligned TM helices driving signaling.”
  • Revises understanding of single-pass RTK signaling: flexibility, not rigidity.