Ligand-receptor interaction on the cell surface has traditionally been analyzed by using radioisotope-labeled ligands. The reaction is carried out at low temperatures such as 4 o C or on ice in order to prevent endocytosis of the ligandreceptor complex, followed by extensive washing to remove unbound ligands from cells. To overcome these drawbacks, in the present study we have employed fluorescence correlation spectroscopy (FCS) with single-molecule sensitivity for the analysis of the interaction of epidermal growth factor (EGF) with its receptor, EGFR, on the surface of living cells, and have successfully determined their dissociation constants. Moreover, FCS has also been used to determine the diffusion coefficient of EGFR in the membrane as well as to quantify the number of EGFR molecules expressed on the cell surface.

Intercellular signals induce various cellular responses, including growth, proliferation, and differentiation, via the dynamic processes of signal transduction pathways. For cell fate decisions, ligand-binding induces the phosphorylation of ERBB receptors, which in turn activate downstream molecules. The ERBB family includes four subtypes, which diverged through two gene duplications from a common ancestor. Differences in the expression patterns of the subtypes have been reported between different organs in the human body. However, how these different expression properties influence the diverse phosphorylation levels of ERBB proteins is not well understood. Here we study the origin of the phosphorylation responses by experimental and mathematical analyses. The experimental measurements clarified that the phosphorylation levels heavily depend on the ERBB expression profiles. We developed a mathematical model consisting of the four subtypes as monomers, homodimers, and heterodimers and estimated the rate constants governing the phosphorylation responses from the experimental data. To understand the origin of the diversity, we analyzed the effects of the expression levels and reaction rates of the ERBB subtypes on the diversity. The difference in phosphorylation rates between ERBB subtypes showed a much greater contribution to the diversity than did the dimerization rates. This result implies that divergent evolution in phosphorylation reactions rather than in dimerization reactions after whole genome duplications was essential for increasing the diversity of the phosphorylation responses.This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

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