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Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer

Schiffer, Hans H. ; Reding, Esther C. ; Fuhs, Stephen R. ; Lu, Qing ; Piu, Fabrice ; Wong, Steven ; Littler, Pey Lih H. ; Weiner, Dave M. ; Keefe, William and Tan, Phil K. , et al. (2007) In Molecular Pharmacology 71(2). p.508-518
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... (More)

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.

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publishing date
type
Contribution to journal
publication status
published
subject
in
Molecular Pharmacology
volume
71
issue
2
pages
508 - 518
publisher
American Society for Pharmacology and Experimental Therapeutics
external identifiers
  • scopus:33846465419
  • pmid:16968809
ISSN
0026-895X
DOI
10.1124/mol.106.027656
language
English
LU publication?
no
id
1a65c146-13f7-40f2-a2a7-2f981e4f86b8
date added to LUP
2019-10-02 10:29:27
date last changed
2020-01-13 02:26:12
@article{1a65c146-13f7-40f2-a2a7-2f981e4f86b8,
  abstract     = {<p>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.</p>},
  author       = {Schiffer, Hans H. and Reding, Esther C. and Fuhs, Stephen R. and Lu, Qing and Piu, Fabrice and Wong, Steven and Littler, Pey Lih H. and Weiner, Dave M. and Keefe, William and Tan, Phil K. and Nash, Norman R. and Knapp, Anne E. and Olsson, Roger and Brann, Mark R.},
  issn         = {0026-895X},
  language     = {eng},
  month        = {02},
  number       = {2},
  pages        = {508--518},
  publisher    = {American Society for Pharmacology and Experimental Therapeutics},
  series       = {Molecular Pharmacology},
  title        = {Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer},
  url          = {http://dx.doi.org/10.1124/mol.106.027656},
  doi          = {10.1124/mol.106.027656},
  volume       = {71},
  year         = {2007},
}