RT Journal Article
SR Electronic
T1 Monitoring Interactions between Receptor Tyrosine Kinases and Their Downstream Effector Proteins in Living Cells Using Bioluminescence Resonance Energy Transfer
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1440
OP 1446
DO 10.1124/mol.107.039636
VO 72
IS 6
A1 Philip K. Tan
A1 Jean Wang
A1 Pey-Lih H. Littler
A1 Kenneth K. Wong
A1 Timothy A. Sweetnam
A1 William Keefe
A1 Norman R. Nash
A1 Esther C. Reding
A1 Fabrice Piu
A1 Mark R. Brann
A1 Hans H. Schiffer
YR 2007
UL http://molpharm.aspetjournals.org/content/72/6/1440.abstract
AB 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), platelet-derived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLCγ1). 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, α polypeptide (PDGFRA) isoforms (V561D; D842V and Δ842–845) 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 structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators. The American Society for Pharmacology and Experimental Therapeutics