Rotational dynamics of the epidermal growth factor receptor.
We have examined the rotational mobility of SL-EGF, a bifunctional adduct of bis(sulfo-N-succinimidyl)-[(15)N,(2)H(16)]-doxyl-2-spiro-4'-pimelate and [Lys3,Tyr22]-murine epidermal growth factor, bound to the EGF receptor in A431 membrane vesicles. The linear EPR spectrum indicated that there was essentially no free SL-EGF in the bound complex preparation. To better define the rotational mobility of the SL-EGF bound to the EGF receptor, ST-EPR spectra were obtained at multiple Zeeman field modulation frequencies. Global analysis with a uniaxial rotational diffusion model of the ST-EPR data yielded two minima that have differences in rotational mobility and in orientation of the SL-EGF relative to the membrane normal axis. The rotational mobilities of the two rotational species are consistent with monomers and dimers or somewhat larger oligomers, such as trimers or tetramers, arguing against a role for higher order receptor clustering in receptor activation. Considering the two minima and previous observations that A431 membrane vesicles contain two distinguishable ligand-binding populations, the ST-EPR spectra were fit with a model having two uniaxial rotating species. This yielded two components that were similar to those obtained from the two original one-component fits, either fast or slow rotational mobility, with different orientations. The model-dependent results obtained suggest that there are potential conformational and rotational differences in the two populations and provide a plausible description for the origin of high- and low-affinity EGF-binding sites that can be tested in future experiments.
Stein
Richard A
RA
Department of Biological Sciences and Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37235.
Hustedt
Eric J
EJ
Staros
James V
JV
Beth
Albert H
AH
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D013113:Spin Labels
D008024:Ligands; D013113:Spin Labels; D004815:Epidermal Growth Factor; D066246:ErbB Receptors