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), 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.
Footnotes
-
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
doi:10.1124/mol.107.039636.
-
ABBREVIATIONS: RTK, receptor tyrosine kinase; BRET, bioluminescence resonance energy transfer; PI3K, phosphatidyl inositol 3-kinase; PLCγ1, phospholipase Cγ1; PKC, protein kinase C; STAT, signal transducer and activator of transcription; Luc, luciferase; GFP, green fluorescence protein; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; EGFR, epidermal growth factor receptor; PDGFRA, platelet-derived growth factor receptor, α polypeptide; PBS, phosphate-buffered saline; BDNF, brain-derived neurotrophic factor; HEK, human embryonic kidney; GPCR, G protein-coupled receptor; FPRL1, formyl peptide receptor-like 1; VEGF, vascular endothelial growth factor; GIST, gastrointestinal stromal tumors; erlotinib, 4-(3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy)quinazoline hydrochloride; imatinib, 4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-[4-(3-pyridyl) pyrimidin-2-ylamino]phenyl]benzamide methanesulfonate; K252a, (+)-10(R)-hydroxy-9(S)-methyl-1-oxo-9,12(R)-epoxy-2,3,9,10,11,12-hexahydro-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester; AG1478, N-(3-chlorophenyl)-N-(6,7-dimethoxyquinazolin-4-yl) amine; PD153035, 4-(3-bromophenylamino)-6,7-dimethoxyquinazoline; PD158780, 4-(3-bromophenylamino)-6-(methylamino)pyrido[3,4-d] pyrimidine; PD168393, N-[4-(3-bromophenylamino)quinazolin-6-yl]-2-propenamide; PD174265, N-[4-(3-bromophenylamino)-6-quinazolinyl]propionamide.
-
↵
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received July 3, 2007.
- Accepted August 10, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|