Epidermal growth factor receptor dimerization monitored in live cells.
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.
Blakely
B T
BT
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5332, USA.
Rossi
F M
FM
Tillotson
B
B
Palmer
M
M
Estelles
A
A
Blau
H M
HM
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
D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D001616:beta-Galactosidase