Enhancement of cell type specificity by quantitative modulation of a chimeric ligand

Abstract

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-α-2a (IFNα-2a) to initiate signal transduction. We first connected EGF and IFNα-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 IFNα-2a portion that progressively decrease both the on rate and the dissociation constant of the IFNα-2a-IFNα receptor 2 (IFNAR2) interaction. Using this strategy, we designed chimeric proteins in which the activation of the IFNα 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 IFNα-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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.