Spatially defined EGF receptor activation reveals an F-actin-dependent phospho-Erk signaling complex.

We investigated the association of signaling proteins with epidermal growth factor (EGF) receptors (EGFR) using biotinylated EGF bound to streptavidin that is covalently coupled in an ordered array of micron-sized features on silicon surfaces. Using NIH-3T3 cells stably expressing EGFR, we observe concentration of fluorescently labeled receptors and stimulated tyrosine phosphorylation that are spatially confined to the regions of immobilized EGF and quantified by cross-correlation analysis. We observe recruitment of phosphorylated paxillin to activated EGFR at these patterned features, as well as β1-containing integrins that preferentially localize to more peripheral EGF features, as quantified by radial fluorescence analysis. In addition, we detect recruitment of EGFP-Ras, MEK, and phosphorylated Erk to patterned EGF in a process that depends on F-actin and phosphoinositides. These studies reveal and quantify the coformation of multiprotein EGFR signaling complexes at the plasma membrane in response to micropatterned growth factors.