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J. Biol. Chem., Vol. 283, Issue 13, 8469-8476, March 28, 2008

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Enhancement of Cell Type Specificity by Quantitative Modulation of a Chimeric Ligand^*

From the Department of Systems Biology, Harvard Medical School, Boston Massachusetts, 02115

Evolution modulates the quantitative characteristics of protein interactions and often uses combinations of weak interactions to achieve a particular specificity. We addressed how quantitative optimization might be used in the design of multidomain proteins, using a chimera containing epidermal growth factor (EGF) as a cell targeting element and interferon--2a (IFN-2a) to initiate signal transduction. We first connected EGF and IFN-2a via a linker that allows both ligands to bind to their receptors on a cell surface and then incorporated a series of mutations into the IFN-2a portion that progressively decrease both the on rate and the dissociation constant of the IFN-2a-IFN receptor 2 (IFNAR2) interaction. Using this strategy, we designed chimeric proteins in which the activation of the IFN receptor in HeLa, A431, and engineered Daudi cells depends on the presence of EGF receptor on the same cell. The mutant chimeric proteins also inhibited proliferation of IFN-sensitive cells in an EGF receptor-dependent manner. These results provide insights into the quantitative requirements for specific binding to multisubunit receptors and illustrate the value of a quantitative approach in the design of synthetic-biological constructs.

Received for publication, October 12, 2007 , and in revised form, January 10, 2008.

^* This work was supported by in part by grants from National Institutes of Health and funds from Harvard University (to P. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures and a supplemental table.

¹ Supported by a Fulbright-M. E. C. (Spain) postdoctoral fellowship.

² To whom correspondence should be addressed: Dept. of Systems Biology, Harvard Medical School, 200 Longwood Ave., Boston MA, 02115. Tel.: 617-432-6401; E-mail: pamela_silver{at}hms.harvard.edu.

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