Rotational Dynamics of the Epidermal Growth Factor Receptor

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• 作者：Richard A. Stein ; Eric J. Hustedt ; James V. Staros ; and Albert H. Beth
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：February 12, 2002
• 年：2002
• 卷：41
• 期：6
• 页码：1957 - 1964
• 全文大小：81K
• 年卷期：v.41,no.6(February 12, 2002)
• ISSN：1520-4995

文摘

We have examined the rotational mobility of SL-EGF, a bifunctional adduct of bis(sulfo-N-succinimidyl)-[¹⁵N,²H₁₆]-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 therewas essentially no free SL-EGF in the bound complex preparation. To better define the rotational mobilityof the SL-EGF bound to the EGF receptor, ST-EPR spectra were obtained at multiple Zeeman fieldmodulation frequencies. Global analysis with a uniaxial rotational diffusion model of the ST-EPR datayielded two minima that have differences in rotational mobility and in orientation of the SL-EGF relativeto the membrane normal axis. The rotational mobilities of the two rotational species are consistent withmonomers and dimers or somewhat larger oligomers, such as trimers or tetramers, arguing against a rolefor higher order receptor clustering in receptor activation. Considering the two minima and previousobservations that A431 membrane vesicles contain two distinguishable ligand-binding populations, theST-EPR spectra were fit with a model having two uniaxial rotating species. This yielded two componentsthat were similar to those obtained from the two original one-component fits, either fast or slow rotationalmobility, with different orientations. The model-dependent results obtained suggest that there are potentialconformational and rotational differences in the two populations and provide a plausible description forthe origin of high- and low-affinity EGF-binding sites that can be tested in future experiments.