Oligomerization-function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy.

• Yamashita, Hirotaka

• Yano, Yoshiaki

• Kawano, Kenichi

• 松崎, 勝巳

抄録

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor-ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag-probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1nM) was lower than that for half-maximum autophosphorylation (~8nM), which suggested the presence of an inactive dimer binding a single EGF molecule.

収録刊行物

• Biochimica et biophysica acta

  Biochimica et biophysica acta 1848 (6), 1359-1366, 2015-03-11

  Elsevier BV

キーワード

• Dimerization
• Live-cell imaging
• FRET

詳細情報

• CRID: 1050001335813492352
• NII論文ID: 120005621952
• NII書誌ID: AA00564635
• ISSN: 00063002
• HANDLE: 2433/198685
• 本文言語コード: en
• 資料種別: journal article
• データソース種別:
  • IRDB
  • CiNii Articles