Received for publication July 5, 2007.
Revised August 10, 2007.
Accepted for publication August 10, 2007.

Monitoring interactions between receptor tyrosine kinases and their downstream effector proteins in living cells using Bioluminescence Resonance Energy Transfer (BRET)

Abstract

A limited number of whole-cell assays are available that allow monitoring receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. Here we present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the RTK super family. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTK's and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for 9 RTK's derived from 4 subfamilies (ErbB, PDGF, TRK, VEGF), monitoring the interactions with 5 effectors (Grb2, p85, Stat5a, Shc46, PLC1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in their carboxy-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 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. 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.

Key words: NGF/EGF, PDGF, Func. analysis receptor/ion channel mutants, Membrane targets