Enhancement of cell type specificity by quantitative modulation of a chimeric ligand.
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-alpha-2a (IFNalpha-2a) to initiate signal transduction. We first connected EGF and IFNalpha-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 IFNalpha-2a portion that progressively decrease both the on rate and the dissociation constant of the IFNalpha-2a-IFNalpha receptor 2 (IFNAR2) interaction. Using this strategy, we designed chimeric proteins in which the activation of the IFNalpha 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 IFNalpha-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.
Cironi
Pablo
P
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Swinburne
Ian A
IA
Silver
Pamela A
PA
D002460:Cell Line; D002468:Cell Physiological Phenomena; D049109:Cell Proliferation; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor / Q000378:metabolism; D012680:Sensitivity and Specificity
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D008024:Ligands; D011994:Recombinant Proteins; D050794:STAT1 Transcription Factor; D004815:Epidermal Growth Factor; D066246:ErbB Receptors