Monitoring Interactions between Receptor Tyrosine Kinases and Their Downstream Effector Proteins in Living Cells Using Bioluminescence Resonance Energy Transfer

Abstract

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...

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation

“…As shown in Fig. 3A, supra maximal activation of EGFR with 100 ng/ml epidermal growth factor (EGF) [38] caused a rapid increase in PtdIns(3,4,5) P 3 reflecting PI3K activation (Fig. 3A second to right panel).…”

Receptor Tyrosine Kinase Signaling

“…Upon co-expression of fusion molecules in live cells, protein-protein interactions or conformational changes can be assessed by measuring the ratio of emissions from the donor and acceptor (reviewed in Siddiqui et al, 2013). One such study utilizing this technology examined the interactions between RTKs of the ErbB, Kit, PDGF, Trk and VEGF receptor families with the signaling molecules Grb2, p85, Stat5a, Shc46 and PLCγ1 in transformed human embryonic kidney cells, revealing specific receptor-signaling molecule interactions in response to growth factor treatment (Tan et al, 2007). Additional studies have employed BRET to examine receptor conformational changes upon ligand treatment.…”

The New Era of Bioluminescence Resonance Energy Transfer Technology

“…RTK) activity in a signaling pathway-specific manner. Tan PK et al [30] presented the general use of the BRET2 technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase super-family. The results presented by this group showed that BRET as a powerful technology to study pharmacology and signaling properties of the RTK super-family in living cells.…”

“…In a recent study, by developing BRET-based cellular RTK assays, it has been shown that discriminating pathway specific signals is important to facilitate the pathway specific drug candidate identification process [30], which will eventually require a HTS screen. Also, a HIV protease based BRET2 biosensor assay was developed [51], which can be adapted for high throughput screening of new HIV-1 protease inhibitors.…”