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Single-Molecule Analysis of Epidermal Growth Factor Signaling that Leads to Ultrasensitive Calcium Response

Takeshi Uyemura ^*, Hiroaki Takagi , Toshio Yanagida  and Yasushi Sako 

^* Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, and Laboratories of Nanobiology, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

Correspondence: Address reprint requests to Yasushi Sako, Laboratories of Nanobiology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka, 565-0871, Japan. Tel.: 81-6-6879-4426; Fax: 81-6-6879-4427; E-mail: sako{at}phys1.med.osaka-u.ac.jp.

Quantitative relationships between inputs and outputs of signaling systems are fundamental information for the understanding of the mechanism of signal transduction. Here we report the correlation between the number of epidermal growth factor (EGF) bindings and the response probability of intracellular calcium elevation. Binding of EGF molecules and changes of intracellular calcium concentration were measured for identical HeLa human epithelial cells. It was found that 300 molecules of EGF were enough to induce calcium response in half of the cells. This number is quite small compared to the number of EGF receptors (EGFR) expressed on the cell surface (50,000). There was a sigmoidal correlation between the response probability and the number of EGF bindings, meaning an ultrasensitive reaction. Analysis of the cluster size distribution of EGF demonstrated that dimerization of EGFR contributes to this switch-like ultrasensitive response. Single-molecule analysis revealed that EGF bound faster to clusters of EGFR than to monomers. This property should be important for effective formation of signaling dimers of EGFR under very small numbers of EGF bindings and suggests that the expression of excess amounts of EGFR on the cell surface is required to prepare predimers of EGFR with a large association rate constant to EGF.

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