Tenascin cytotactin epidermal growth factor-like repeat binds epidermal growth factor receptor with low affinity

Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin (tenascin C) can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of classical growth factors such as EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound 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 a significantly higher mobility in Ten14-EGFR complex compared to that of the EGF-EGFR complex; we hypothesized this may be attributed 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 74 microM 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 so as to induce degradation of receptor, or undergo EGFR-mediated internalization over either the short (20 min) or long (48 h) term. This transient interaction 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.