Luciferase fragment complementation imaging of conformational changes in the epidermal growth factor receptor.
Crystal structures of the epidermal growth factor (EGF) receptor suggest that its activation is associated with extensive conformational changes in both the extracellular and intracellular domains. However, evidence of these structural dynamics in intact cells has been lacking. Here we use luciferase complementation imaging to follow EGF-induced conformational changes in its receptor in real time in live cells. When the luciferase fragments are fused to the C terminus of an EGF receptor lacking the cytoplasmic domain, EGF stimulates a rapid increase in luciferase activity, consistent with ligand-induced receptor dimerization. However, when the luciferase fragments are fused to the C terminus of the full-length receptor, EGF induces a rapid but transient decrease in luciferase activity. The decrease requires tyrosine kinase activity, whereas the subsequent recovery requires MAP kinase activity. Our data demonstrate the utility of the luciferase system for in vivo imaging changes in EGF receptor dimerization and conformation. They also identify two sequential ligand-induced conformational changes in the EGF receptor.
Yang
Katherine S
KS
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Ilagan
Ma Xenia G
MX
Piwnica-Worms
David
D
Pike
Linda J
LJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D008156:Luciferases; D066246:ErbB Receptors; D020929:Mitogen-Activated Protein Kinase Kinases