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ACADIA Pharmaceuticals, Inc., San Diego, California
Received July 3, 2007; accepted August 10, 2007
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Abstract |
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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 structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators.
). 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 assays (Olive, 2004; Minor, 2005). A shift toward the use of more whole-cell-based RTK assays is expected to lead to a better prediction of the clinical outcome of new drug candidates. Furthermore, cancer drugs are increasingly designed to target specific cell-signaling pathways, because cancer types show signaling pathway-specific deregulation signatures (Bild et al., 2006). The development of whole-cell-based RTK assays, which allow discriminating pathway specific signals, is important to facilitate this process.
We used the bioluminescence resonance energy transfer (BRET) technology (Xu et al., 1999; Angers et al., 2000; Pfleger and Eidne, 2006) and developed new whole-cell receptor tyrosine kinase assays, which enabled us to monitor in living cells the ligand-induced recruitment of downstream effector proteins to various members of the RTK superfamily. Many of the RTK-effector protein interactions depend on the autophosphorylation of specific tyrosine residues in the intracellular carboxyl terminus of an RTK. They control the assembly of larger protein complexes that are involved in building, shaping, and directing specific RTK signaling pathways (illustrated in Fig. 1a) (Schlessinger, 2000). We included in our study RTK effector proteins from different signaling pathways: the adapter proteins Grb2 and Shc46 in the Ras/mitogen-activated protein kinase pathway; p85, the regulatory subunit of phosphatidyl inositol 3-kinase (PI3K) in the PI3K/Akt pathway; phospholipase C1 (PLC1) in the PLC1/PKC pathway; and the Stat5a protein in the STAT pathways. The central role of protein-protein interactions for RTK activation and signaling makes the BRET technology a method of choice to study RTK function in living cells in a signaling pathway-specific modus. RTK BRET-2 assays are highly sensitive and precisely dissect and quantify the pharmacological responses and signaling properties of RTKs. Earlier BRET studies analyzed the interactions of the insulin receptor with the insulin receptor substrate-1 (Blanquart et al., 2006), protein tyrosine phosphatase-1B (Blanquart et al., 2005), or the adapter protein Grb14 (Nouaille et al., 2006a,b). We demonstrate here that the BRET technology is universally applicable to the entire RTK superfamily and discuss the advantages of this technology compared with other methods that measure RTK activity.
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Materials and Methods |
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). The following DNA amounts were used for the RTK-BRET-2 assays: 1 µg of RTK-Luc DNA and 20 µg of GFP2-effector DNA, except where noted. The GPCR FPRL1 BRET-2 assay was performed by cotransfecting 1 µg of FPRL1-Luc DNA and 20 µg of GFP2-BA2 DNA. The ratio of 1:20 was predetermined in saturation experiments to be optimal for obtaining the best ligand induced increase in the BRET-2 signals. These DNAs in water were mixed with 80 µl of 2.5 M CaCl2 and 1.7 ml of 2x HBS (50 mM HEPES, pH 7.05, 280 mM NaCl, and 1.5 mM Na2HPO4) and incubated at room temperature for 3 to 5 min. The media was removed from the cells, and 10 ml of fresh media was added to the transfection mixture, which was then transferred onto the cells. After 2 to4hof incubation at 37°C, the medium was replaced with 10 ml of fresh media. On the next day, the cells were serum starved by replacing the media with 10 ml of Dulbecco's modified Eagle's medium supplemented with 0.1% fetal bovine serum and 1x penicillin-streptomycin-L-glutamine for 16 to 20 h before harvesting.
Plasmids. BRET-2 vectors expressing Renilla reniformis luciferase (pRluc-N) and green fluorescent protein 2 (pGFP2-N and pGFP2-C) were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Human cDNAs encoding RTKs or RTK effector proteins were obtained by standard reverse transcription-polymerase chain reaction on poly-A-RNA isolated from various human tissues or human cell lines. Genes were amplified without a stop codon when appropriate and subcloned in frame into the BRET-2 vectors. For expressing the amino- or carboxyl-terminally GFP2-tagged tandem SH2 domains as fusion protein SH2(PLC1)-GFP2 or GFP2-SH2(PLC1), the bases encoding amino acids 507 to 790 of human PLC1 were polymerase chain reaction-amplified from GFP2-PLC1 cDNA and inserted into pGFP2-N and pGFP2-C. Epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, polypeptide (PDGFRA) mutants were produced by standard site-directed mutagenesis methods from EGFR-Luc and PDGFRA-Luc, respectively. Accuracy of the sequences of all constructs used in this study has been confirmed.
RTK BRET-2 Measurement. Transfected cells were rinsed once with Dulbecco's PBS, detached with Dulbecco's PBS containing 5 mM EDTA, and resuspended at 4 to 8 x 106 cells/ml in BRET buffer (PBS containing 0.1% D-glucose and 1 mM sodium pyruvate). All ligand serial dilutions were prepared in BRET buffer containing 0.1% BSA. For agonist assays, 50 µl of 3-fold concentrated ligand dilutions were dispensed into wells of white, flat-bottomed, 96-well plates (Costar; Corning Life Sciences, Acton, MA). For antagonist assays, 25 µl of 6-fold concentrated agonist and 25 µl of 6-fold concentrated antagonist were added together per well. Ligands were incubated for 10 to 20 min with 50 µl of cell suspension to stimulate the interaction of RTK-Luc (bioluminescence donor) with GFP2-tagged downstream effector (fluorescence acceptor). The BRET-2 signal was detected directly after injecting 50 µl of 15 µM coelenterazine 400A (DeepBlueC; PerkinElmer Life and Analytical Sciences) diluted in PBS per well using the POLARstar OPTIMA plate reader (BMG Labtech GmbH, Offenburg, Germany) or the Mithras LB 940 plate reader (Berthold Technologies, Bad Wildbad, Germany). After1sof plate-shaking, luminescence emissions for R. reniformis luciferase and GFP2 were recorded through BRET-optimized filters (luciferase peakm 410 nm; GFP2 peak, 515 nm) for 1 to 2 s in well mode. The time from adding coelenterazine 400A to the plate well until the reading start was sufficient to fully activate luciferase (data not shown). The BRET-2 signal was calculated as the ratio between the luciferase and the GFP2 emission corrected by the background emission of cells transfected with RTK-Luc alone. Nonlinear regression analysis was performed with the software Prism (GraphPad Software Inc., San Diego, CA) to obtain dose response curves and IC50/EC50 values. Throughout the text, EC50 and IC50 values are expressed as pEC50 or pIC50 [molar] values, which are calculated as -log10 of the EC50 or IC50 [molar]. Experiments were repeated two to three times with each data point, performed in triplicate, and expressed as mean ± S.E.M. The ligands EGF, heregulin-
1, platelet-derived growth factor BB (PDGF-BB), and brain-derived neurotrophic factor (BDNF) were purchased from Peprotech (Rocky Hill, NJ), VEGF-C was purchased from R&D Systems (Minneapolis, MN), and WKYMVm was purchased from Tocris Cookson Inc. (Ellisville, MO). Erlotinib (Tarceva) and imatinib (Gleevec) were synthesized by ACADIA Pharmaceuticals, Inc. (San Diego, CA).
Immunoblotting. Transfected cells in BRET buffer were incubated without or with EGF for 10 min and then lysed by adding a 10-fold volume of protein sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.005% bromphenol blue, 5% -mercaptoethanol, and 1 mM sodium orthovanadate). Lysates were electrophoresed through 10% polyacrylamide gels and transferred to nitrocellulose for Western blotting. Luciferase- or GFP2-tagged fusion proteins were detected using monoclonal luciferase antibody 4410 (Chemicon, Temecula, CA) or polyclonal GFP antibody (Cell Signaling Technology, Danvers, MA). Proteins carrying phosphotyrosine residues were detected using the monoclonal antibody 4G10 (Upstate, Charlottesville, VA). Horseradish peroxidase-conjugated secondary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), and SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) were used for developing Western blots.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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First, we applied the BRET technology to study the interaction between the most extensively studied RTK, the EGFR, and the effector protein Grb2 (Fig. 1c). EGFR-Luc and aminoterminal GFP2-tagged Grb2 (GFP2-Grb2) were transiently coexpressed in HEK293T cells. After a 10-min incubation of the cells with 16.7 nM EGF, we detected a 3-fold increase in the BRET-2 ratio from 0.21 ± 0.02 to 0.59 ± 0.04, indicating EGFR activation and recruitment of GFP2-Grb2 (Fig. 1c). Cotreatment of EGF with 3.3 µM erlotinib, an EGFR inhibitor, completely reversed the EGF-induced increase in the BRET-2 ratio (Fig. 1c). Furthermore, these cells yielded a BRET-2 ratio (0.16 ± 0.005) slightly lower than that obtained for untreated cells (0.21 ± 0.02), suggesting a low level of constitutive EGFR activity in untreated cells (discussed below). Similar results were also obtained for the EGFR tyrosine kinase inhibitors AG1478, PD153035, PD158780, PD168393, and PD174265 (data not shown).
The RTK BRET-2 assay responses are based on specific ligand-induced RTK effector interactions. To demonstrate ligand specificity, we tested the peptide WKYMVm, an agonist for the G protein-coupled receptor (GPCR) FPRL1 (formyl peptide receptor-like 1) in the EGFR/Grb2 BRET-2 assay (Fig. 1c). WKYMVm did not produce a response in the EGFR/Grb2 BRET-2 assay but did stimulate a 7-fold response in the FPRL1/BA2 BRET-2 assay (no ligand, 0.04 ± 0.002; WKYMVm, 0.30 ± 0.07), which is monitoring the interaction between the luciferase-tagged FPRL1 and GFP2-tagged -arrestin-2 (BA2) protein (Fig. 1d). As expected, neither EGF nor erlotinib affected the WKYMVm-induced BRET-2 ratio in the FPRL1/BA2 BRET-2 assay (Fig. 1d). To demonstrate that the observed ligand-induced BRET-2 responses were based on specific protein interactions, we coexpressed EGFR-Luc with GFP2-BA2, or FPRL1-Luc with GFP2-Grb2, and stimulated the cells with EGF or WKYMVm, respectively. Neither ligand induced a response in these BRET assays, because the coexpressed receptors and effectors do not specifically interact in vivo (Fig. 1, c and d).
We next tested other EGFR effectors in EGFR BRET-2 assays and quantitatively studied the pharmacological properties of EGFR. The GFP2-tagged effector proteins Grb2, Shc46, p85, PLC1, and Stat5a were individually coexpressed with EGFR-Luc in HEK293T cells, and their BRET-2 responses were detected after incubation of these cells with variable EGF concentrations. We observed a dose-dependent increase in the BRET-2 signal in all EGFR BRET-2 assays that was efficiently inhibited with the EGFR inhibitor erlotinib in a dose-dependent manner (Fig. 2, a–e, and Table 1). Our results show that the different EGFR BRET-2 assays are highly sensitive in detecting responses to the native EGFR agonist EGF, with EC50 values ranging from 30 to 80 pM (summarized in Table 1). It is noteworthy that EGFR-Luc showed significant levels of constitutive activity in the interaction with the downstream effectors Grb2, Shc46, and p85, as indicated by the significantly higher baselines before erlotinib inhibition (Fig. 2, a–c). We previously confirmed that these differences are due to variable levels of constitutive wild-type EGFR activity in the different EGFR signaling transduction pathways (Schiffer et al., 2007). Our results presented in Fig. 2, d and e, suggest that there might be also a low level of constitutive EGFR activity in the Stat5a and PLC1/PKC pathways, but this has not been further explored or confirmed. Each of the presented EGFR BRET-2 assays monitors only one specific receptor-protein interaction that is involved in activating/modulating one specific downstream signaling pathway. Thus, these EGFR BRET-2 assays represent signaling pathway-specific, whole-cell based assays.
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RTK BRET-2 Assays Are Dependent on Autophosphorylation of Specific Tyrosine Residues. Phosphorylated tyrosine residues localized in the intracellular carboxyl terminus of EGFR (Heldin, 1995) and specific phosphotyrosine binding or SH2 domains in the effector proteins (Schlessinger and Lemmon, 2003) mediate all the EGFR effector interactions we studied in Fig. 2. EGFR tyrosines 1068, 1086, 1101, 1114, 1148, and 1173 are involved in direct or indirect binding of the effector Grb2 (Schulze et al., 2005). We mutated these tyrosine residues to phenylalanine to verify that the EGFR/Grb2 BRET-2 signal is dependent on their phosphorylation. Introducing all six Tyr-to-Phe alterations into EGFR-Luc abolished the EGF-induced BRET-2/Grb2 response by 90 ± 0.9% compared with wild-type EGFR-Luc (Fig. 2f). We observed 66 ± 0.9% and 42 ± 1.0% impairment of the BRET-2/Grb2 responses for EGFR-Luc isoforms carrying five (Y1068F, Y1086F, Y1101F, Y1114F, Y1173F) or four (Y1086F, Y1101F, Y1114F, Y1173F) of the six Tyr-to-Phe changes, respectively (Fig. 2f). Three Tyr-to-Phe changes (Y1086F, Y1101F, Y1173F) caused only a 16 ± 1.1% inhibition of the BRET-2/Grb2 response (Fig. 2f). In contrast to the results from the BRET-2/Grb2 assays, abolishing phosphorylation at the six tyrosine residues only partially affected EGF-induced responses in the EGFR BRET-2/p85/STAT5a or /PLC1 assays (data not shown). Consistent with our results, a kinase-deficient EGFR-Luc isoform carrying the kinase domain mutation K721M completely abolished all BRET-2 responses with the effector proteins tested (data not shown). Finally, we performed EGFR BRET-2 assays using the two tandem repeat SH2 domains of PLC1 as the effector and found that the phospho-tyrosine binding domains of the effector PLC1 were sufficient for generating an EGFR BRET-2 response (Supplemental Fig. 2). Our experiments show that the EGFR BRET-2 assays are mediated by interactions between specific autophosphorylated tyrosine residues on activated EGFR and specific phosphotyrosine binding domains in RTK effectors. Furthermore, we demonstrated the sensitivity, reproducibility, and robustness of the RTK BRET-2 assay, which makes it an ideal tool to detect small functional differences in structure-function studies. It has been reported that the kinase domain of activated RTKs also trans-phosphorylates tyrosines on recruited effectors such as Grb2 and PLC1 (Schlessinger, 2000). The results from our EGFR-BRET-2 assays, therefore, suggest that tyrosine phosphorylation in the GFP2-tagged effectors should increase upon ligand treatment. Western blotting of lysates from EGFR BRET-2 assay cells confirmed this prediction (Supplemental Fig. 3).
Enabling the RTK Superfamily in BRET-2 Assays. Fifty-eight human RTKs have been described in the human genome (Robinson et al., 2000). We tested whether the RTK BRET-2 assay was applicable to study the pharmacological and signaling properties of RTKs other than EGFR. Indeed, we were able to measure RTK activation of other members of the RTK superfamily in BRET-2 assays (Fig. 3, a–d). Heregulin-1 stimulated GFP2-Grb2 recruitment to ErbB4-Luc, another member of the EGFR family of growth factor receptors (Fig. 3a). PDGF-BB stimulated a dose-dependent increase of the BRET-2 ratio in a platelet-derived growth factor receptor, polypeptide BRET-2/Grb2 assay (Fig. 3b). BDNF stimulated the neurotrophic tyrosine kinase receptor (Trk) family member TrkB in a TrkB/Shc46 BRET-2 assay (Fig. 3c). VEGF-C activated VEGF receptor 3 in a BRET-2/Grb2 assay (Fig. 3d). We enabled additional RTKs from these subfamilies in RTK BRET-2 assays (Table 1). Most of the RTKs analyzed in this study did interact with multiple effector proteins, which correlated well with published results about their signal transduction (Table 1). The sensitivity of all RTK BRET-2 assays for activation by their endogenous in vivo ligands was in the nanomolar range, which is in agreement with results from other methods and reflects the high in vivo potency of these growth factors. We have also shown that tyrosine kinase inhibitors with specificity for the tested RTKs efficiently inhibit their agonist-induced BRET-2 responses (summarized in Table 1). All together, we demonstrate that the RTK BRET-2 technology can measure activity of members from four subfamilies of RTKs. Therefore, it is possible that the BRET-2 technology could be used to monitor the activity of most, if not all, known RTKs.
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,b). We studied two mutations causing a nonsynonymous amino acid alterations (V561D and D842V) and a four amino acid deletion mutation 842–845 with PDGFRA BRET-2 assays (Fig. 3e). In the absence of the agonist PDGF-BB, all mutant PDGFRA-Luc isoforms exhibited higher BRET-2 ratios compared with wild-type PDGFRA-Luc (Fig. 3e, no ligand), which is indicative of constitutive activity in the mutant isoforms. Addition of PDGF-BB produced a dose-dependent increase of the BRET-2 ratio for the wild-type receptor, which reaches a maximum at the level of the untreated mutated PDGFRA-Luc isoforms. The BRET-2 ratios of all PDGFRA-Luc isoforms remained unchanged in the presence of PDGF-BB (Fig. 3e). These findings suggest that the mutant PDGFRA-Luc isoforms are fully constitutively activated and ligand-insensitive receptors. Although, all three mutant isoforms show similar levels of constitutive activity, they showed different sensitivities to the PDGFRA inhibitor imatinib (Gleevec). Imatinib reduced the PDGF-BB-induced BRET-2 ratio of wild-type PDGFRA-Luc with a pIC50 = 6.62 ± 0.12 M. The constitutive activity of the mutant isoforms V561D and 842–845, in the absence of PDGF-BB, was inhibited by imatinib with similar potencies (V561D pIC50 = 6.98 ± 0.13 M and 842–845 pIC50 = 7.15 ± 0.10 M), whereas the mutant D842V isoform was approximately 30-fold less sensitive to imatinib (D842V pIC50 = 5.13 ± 0.10 M) (Fig. 3f). These results demonstrate that the RTK BRET-2 assays can be used to quantitatively characterize the signaling properties and pharmacology of constitutively active wild-type and mutant RTK isoforms, which is important for identifying the signaling pathways, principally driving a cancer pathogenesis and the design of treatment strategies.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The high sensitivity of the RTK BRET assay precisely dissects and characterizes RTK structure-function relationships. We confirmed and quantified the contribution of 6 EGFR tyrosine residues in the recruitment of the adapter protein Grb2 (Fig. 3). Furthermore, we demonstrated that different mutant PDGFRA isoforms identified in GIST are fully constitutively active but show different sensitivities to the inhibitor imatinib. These results were previously not detected in experiments using phosphotyrosine antibodies. RTK BRET-2 assays may be useful in the future to dissect the pharmacological properties of somatic mutations in oncogenic RTKs and help to define better treatment strategies in cancer. In conclusion, RTK BRET assays represent a novel assay platform that will facilitate the characterization of RTK pharmacology and signaling and strengthen ongoing research efforts to identify and develop novel drugs targeting RTKs. |
Acknowledgements |
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Footnotes |
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ABBREVIATIONS: RTK, receptor tyrosine kinase; BRET, bioluminescence resonance energy transfer; PI3K, phosphatidyl inositol 3-kinase; PLC1, phospholipase C1; 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, 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. 
Address correspondence to: Hans H. Schiffer, ACADIA Pharmaceuticals, 3911 Sorrento Valley Blvd., San Diego, CA 92121. E-mail: hschiffer{at}acadiapharm.com
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References |
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). The EGFR tyrosine kinase inhibitor erlotinib efficiently inhibited the observed EGF-stimulated BRET-2 responses (
; a–e). In these antagonist assays, cells were incubated for 10 min in the presence of 16.7 nM EGF and increasing concentrations of erlotinib. We observed constitutive wild-type EGFR activity mainly in the EGFR Grb2/shc/p85 BRET-2 assays (previously confirmed by Schiffer et al., 2007