Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response
Takeshi Uyemura 1, Hiroaki Takagi 1, Toshio Yanagida 1 and Yasushi Sako 1*
1 Osaka University
* To whom correspondence should be addressed. E-mail: sako{at}phys1.med.osaka-u.ac.jp.
Submitted on October 5, 2004
Revised on November 19, 2004
Accepted on 22 February 2005
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Abstract |
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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 the half of 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 singaling 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 high association rate constant to EGF.
Key Words:
calcium response, epidermal growth factor, signal transduction, single molecule imaging
