Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14 th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound

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... structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits significantly higher mobility in Ten14-EGFR complex compared to the EGF-EGFR complex; we attributed this to loss of key high-affinity interactions within the Ten14-EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant K D of 74µM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability to induce degradation of receptor, or undergo EGFR-mediated internalization. This transient interaction of Ten14 with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling. iv We investigated whether Ten14-mediated surface restriction of EGFR resulted in altered cellular responses compared to EGF. Activation of PLCγ and m-calpain, molecules associated with migration, were noted even at sub-saturating doses of Ten14. However, activation of ERK/MAPK, p90RSK and Elk1, factors affecting proliferation, remained low even at high Ten14 concentrations. Similar activation profiles were observed for EGF-treated cells at 4 o C, a maneuver that limits receptor internalization. We demonstrated a direct concurrent effect of such altered signaling on overall biophysical responses -sustained migration was observed at lower levels of Ten14 that activated PLCγ, but proliferation remained basal. We present a novel class of EGFR ligands that can potentially signal as a part of the matrix, triggering select signaling cascades leading to a directed cellular response from an otherwise pleiotropic receptor.