A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), plateletderived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLC␥1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, ␣ polypeptide (PDGFRA) isoforms (V561D; D842V and ⌬842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structurefunction and will facilitate drug discovery efforts for the identification of novel RTK modulators.Receptor tyrosine kinases (RTKs) represent a broad class of cell surface receptors that transduce signals across the cell membrane and regulate cell proliferation, survival, differentiation and migration (Schlessinger, 2000). Activation or overexpression of most of the known RTKs has been linked to some form of cancer (Sawyer et al., 2003;Krause and Van Etten, 2005). Although the RTKs are prime targets for treatment of cancer, only a small number of therapeutics has been identified despite massive drug discovery efforts. Many novel cancer drugs show only a limited response rate and cannot be applied to treat a wide spectrum of cancer types (Sawyers, 2004;Pao and Miller, 2005). One possible reason for these outcomes has been that the majority of methods used to identify kinase inhibitors are biochemical tyrosine kinase Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.039636.□ S The online version of this article (available at http://molpharm. aspetjour...

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 [pEC 50 of epidermal growth factor (EGF) ϭ 10.1 Ϯ 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 ligandinduced interaction between EGFR and signaling pathwayspecifying adapter proteins.Overexpression and activation of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), plays an important role in the etiology of non-small-cell lung cancer (NSCLC) . Therefore, EGFR is recognized as a key target for the development of NSCLC therapies (Hynes and Lane, 2005). Two drug development strategies focusing on EGFR inhibition are currently pursued: 1) the identification of reversible or irreversible small molecule drugs that inhibit the intracellular tyrosine kinase activity of EGFR by competitively binding to the ATP-binding site of the kinase domain, and 2) the identification of humanized monoclonal antibodies (mAbs) that interact with extracellular EGFR domains interfering with ligand binding (e.g., epidermal growth factor; EGF) or EGFR dimerization (Hynes and Lane, 2005). The reversible small molecule EGFR inhibitors Iressa (gefitinib) (Herbst et al., 2004) and Tarceva (erlotinib) (Minna and Dowell, 2005) and the antibody drug Erbitux (cetuximab; IMC-C225) (Goldberg, 2005) have already been marketed in the United States for NSCLC. However, early clinical studies observed only a 10 to 15% response rate in a U.S. population of unselected NSCLC Article, publication date, and c...

No abstract

No abstract