Abstract
We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing β-galactosidase (β-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as β-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. β-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and β-gal complementation. These results demonstrate that β-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.
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Acknowledgements
We thank Xiao-Jia Chang, Carol Zuschlag, Carol Charlton, Joyce Tung (funded by Howard Hughes Medical Institute undergraduate research fellowship), and Patricia Wong (funded by the Stanford Medical School Alumni Scholarship) for technical assistance. F.M.V.R. was funded by a fellowship from the Human Frontier Science Program. This research was supported by National Institute of Health grants to H.M.B.
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Blakely, B., Rossi, F., Tillotson, B. et al. Epidermal growth factor receptor dimerization monitored in live cells. Nat Biotechnol 18, 218–222 (2000). https://doi.org/10.1038/72686
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DOI: https://doi.org/10.1038/72686
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