Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor.
We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to fluorescence resonance energy homo-transfer. Using time-resolved fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.
Whitson
Kristin B
KB
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.
Beechem
Joseph M
JM
Beth
Albert H
AH
Staros
James V
JV
D000818:Animals; D019281:Dimerization; D004735:Energy Transfer; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D008081:Liposomes; D051379:Mice; D009930:Organic Chemicals; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
C417664:Alexa594; D005456:Fluorescent Dyes; D008081:Liposomes; D009930:Organic Chemicals; D004815:Epidermal Growth Factor; D066246:ErbB Receptors