Epidermal growth factor receptor dimerization monitored in live cells |
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Authors: | Blakely B T Rossi F M Tillotson B Palmer M Estelles A Blau H M |
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Institution: | Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5332, USA. |
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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 beta-galactosidase (beta-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 beta-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. beta-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and beta-gal complementation. These results demonstrate that beta-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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