title: Epidermal growth factor receptor dimerization monitored in live cells.
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.
authors.affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5332, USA.
authors.forename: B T
authors.initials: BT
authors.lastname: Blakely
authors.forename: F M
authors.initials: FM
authors.lastname: Rossi
authors.forename: B
authors.initials: B
authors.lastname: Tillotson
authors.forename: M
authors.initials: M
authors.lastname: Palmer
authors.forename: A
authors.initials: A
authors.lastname: Estelles
authors.forename: H M
authors.initials: HM
authors.lastname: Blau
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D001616:beta-Galactosidase