title: Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer.
abstract: We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.1+/-0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligand-induced interaction between EGFR and signaling pathway-specifying adapter proteins.
authors.affiliation: ACADIA Pharmaceuticals, 3911 Sorrento Valley Blvd., San Diego, CA 92121, USA. hschiffer@acadia-pharm.com
authors.forename: Hans H
authors.initials: HH
authors.lastname: Schiffer
authors.forename: Esther C
authors.initials: EC
authors.lastname: Reding
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Fuhs
authors.forename: Qing
authors.initials: Q
authors.lastname: Lu
authors.forename: Fabrice
authors.initials: F
authors.lastname: Piu
authors.forename: Steven
authors.initials: S
authors.lastname: Wong
authors.forename: Pey-Lih H
authors.initials: PL
authors.lastname: Littler
authors.forename: Dave M
authors.initials: DM
authors.lastname: Weiner
authors.forename: William
authors.initials: W
authors.lastname: Keefe
authors.forename: Phil K
authors.initials: PK
authors.lastname: Tan
authors.forename: Norman R
authors.initials: NR
authors.lastname: Nash
authors.forename: Anne E
authors.initials: AE
authors.lastname: Knapp
authors.forename: Roger
authors.initials: R
authors.lastname: Olsson
authors.forename: Mark R
authors.initials: MR
authors.lastname: Brann
mesh_terms: D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins; D008175:Lung Neoplasms / Q000235:genetics; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D020033:Protein Isoforms; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D008164:Luminescent Proteins; D020033:Protein Isoforms; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt