Abstract
Aim:
To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor (EGFR).
Methods:
Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor (EGF) was used to visualize the internalized receptor-ligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated, optimized, and applied to a large-scale screening of a library containing 48 000 synthetic compounds.
Results:
The competition between fluorescent EGF and unlabeled EGF reveals the IC50 of unlabeled EGF is approximately 0.2 nmol/L, which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified. One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase (ERK).
Conclusion:
This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.
Similar content being viewed by others
Article PDF
References
Cohen S . Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem 1962; 237: 1555–62.
Cohen S . The stimulation of epidermal proliferation by a specific protein (EGF). Dev Biol 1965; 12: 394–407.
Nishida T, Nakamura M, Murakami J, Mishima H, Otori T . Epidermal growth factor stimulates corneal epithelial cell attachment to fibronectin through a fibronectin receptor system. Invest Ophthalmol Vis Sci 1992; 33: 2464–9.
Maldonado BA, Furcht LT . Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide. Invest Ophthalmol Vis Sci 1995; 36: 2120–6.
Seger R, Yarden Y, Kashles O, Goldblatt D, Schlessinger J, Shaltiel S . The epidermal growth factor receptor as a substrate for a kinase-splitting membranal proteinase. J Biol Chem 1988; 263: 3496–500.
Yarden Y, Ullrich A . Molecular analysis of signal transduction by growth factors. Biochemistry 1988; 27: 3113–9.
Johnson GL, Lapadat R . Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002; 298: 1911–2.
Widmann C, Gibson S, Jarpe MB, Johnson GL . Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 1999; 79: 143–80.
Ullrich A, Schlessinger J . Signal transduction by receptors with tyrosine kinase activity. Cell 1990; 61: 203–12.
Schlessinger J . Cell signaling by receptor tyrosine kinases. Cell 2000; 103: 211–25.
Weiner HL, Zagzag D . Growth factor receptor tyrosine kinases: cell adhesion kinase family suggests a novel signaling mechanism in cancer. Cancer Invest 2000; 18: 544–54.
Aaronson SA . Growth factors and cancer. Science 1991; 254: 1146–53.
Voldborg BR, Damstrup L, Spang-Thomsen M, Poulsen HS . Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol 1997; 8: 1197–206.
Knorr M, Schuller S, Steuhl KP, Thiel HJ . EGF in therapy of corneal diseases. Principles and possible uses. Ophthalmologe 1992; 89: 119–27 ( in German).
Tarnawski AS, Jones MK . The role of epidermal growth factor (EGF) and its receptor in mucosal protection, adaptation to injury, and ulcer healing: involvement of EGF-R signal transduction pathways. J Clin Gastroenterol 1998; 27 ( Suppl 1): S12–20.
Qian J, Voorbach MJ, Huth JR, Coen ML, Zhang H, Ng SC, et al. Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms. Anal Biochem 2004; 328: 131–8.
Dagogo-Jack S, Atkinson S, Kendall-Taylor P . Homologous radioimmunoassay for epidermal growth factor in human saliva. J Immunoassay 1985; 6: 125–36.
Zhang JH, Chung TD, Oldenburg KR . A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999; 4: 67–73.
Ghosh RN, DeBiasio R, Hudson CC, Ramer ER, Cowan CL, Oakley RH . Quantitative cell-based high-content screening for vaso-pressin receptor agonists using transfluor technology. J Biomol Screen 2005; 10: 476–84.
Osherov N, Gazit A, Gilon C, Levitzki A . Selective inhibition of the epidermal growth factor and HER2/neu receptors by tyrphostins. J Biol Chem 1993; 268: 11 134–42.
Ding GJ, Fischer PA, Boltz RC, Schmidt JA, Colaianne JJ, Gough A, et al. Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Development and use of a high capacity fluorescence cytometric system. J Biol Chem 1998; 273: 28 897–905.
Haskins JR, Rowse P, Rahbari R, de la Iglesia FA . Thiazolidinedione toxicity to isolated hepatocytes revealed by coherent multiprobe fluorescence microscopy and correlated with multiparameter flow cytometry of peripheral leukocytes. Arch Toxicol 2001; 75: 425–38.
Plymale DR, Haskins JR, de la Iglesia FA . Monitoring simultaneous subcellular events in vitro by means of coherent multiprobe fluorescence. Nat Med 1999; 5: 351–5.
Conway BR, Minor LK, Xu JZ, Gunnet JW, DeBiasio R, D'Andrea MR, et al. Quantification of G-protein coupled receptor inter-nalization using G-protein coupled receptor-green fluorescent protein conjugates with the ArrayScan®trade mark high-content screening system. J Biomol Screen 1999; 4: 75–86.
Author information
Authors and Affiliations
Corresponding author
Additional information
Project supported by grants from the National Natural Sciences Foundation of China (30623008) and Shanghai Commission of Science and Technology (No 04ZR14032, 05DZ22914).
Rights and permissions
About this article
Cite this article
Wang, J., Xie, X. Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators. Acta Pharmacol Sin 28, 1698–1704 (2007). https://doi.org/10.1111/j.1745-7254.2007.00640.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2007.00640.x