Cell surface restriction of EGFR by a tenascin cytotactin-encoded EGF-like repeat is preferential for motility-related signaling.

Many cell surface receptors elicit pleiotropic cellular responses when activated, although some of these responses might be mutually exclusive in any given or at a given time point. One prime example involves the epidermal growth factor receptor (EGFR), which upon ligandation, triggers cell migration and proliferation, two responses that do not occur simultaneously (Wells, 1999). How a cell distinguishes between these two outcomes likely involves differential activation of the myriad of intracellular signaling pathways that are activated by this receptor (Bhalla and Iyengar, 1999). We have demonstrated previously that EGFR-mediated migration and proliferation are distinct cell responses that negatively impact each other; that is, when cells are driven to migrate, the fraction of the cell population undergoing proliferating diminishes (Chen et al., 1994a, 1996b). EGFR-induced motility requires the activation of phospholipase C-γ (PLCγ) (Chen et al., 1996a,b; Polk, 1998), whose activation negatively impacts EGFR-mediated cell proliferation. Interestingly, both proliferation and migration are downstream of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK); however, motility requires ERK to be activated at the plasma membrane (Glading et al., 2001), while proliferation can be triggered by ERK at either the plasma membrane or from intracellular sites (Chen et al., 1994a; Wang et al., 2002). Additionally, cell proliferation requires ERK translocation into the nucleus along with other transcription factors such as Ets-related protein ELK1 and the 90-kDa ribosome S6 protein kinase (p90RSK) that are activated by intracellular ERK (Ebisuya et al., 2005; Rocks et al., 2006). Thus, cellular responses mediated by EGFR signaling appear to be triggered independently by the spatial separation of these key molecules. This mode of signal control adds an important dimension to controlling biophysical responses downstream of EGFR (Burke et al., 2001; Di Fiore and De Camilli, 2001; Ebisuya et al., 2005; Rocks et al., 2006). EGFR is activated by soluble growth factors such as EGF, TGFα, amphiregulin, heparin-binding EGF, betacellulin and a few virally encoded factors (Citri and Yarden, 2006). These peptides bind with very high affinity and lead to internalization of EGFR, activating both motogenic and mitogenic cascades in the process (Wells, 2000). Ligand binding is key to internalization of EGFR—unliganded EGFR fail to internalize and activate downstream molecules from such locales (Haugh et al., 1999b). This led us to speculate that a ligand that limits EGFR activity in a manner so as to trigger signaling selectively from the cell surface might elicit a different spectrum of responses than the classical soluble EGFR ligands such as EGF and TGFα. We and others recently demonstrated that a new class of ligands for EGFR—EGF-like (EGFL) repeats—can activate EGFR with binding modes qualitatively distinct from the classical soluble peptide ligands for EGFR (Swindle et al., 2001; Schenk et al., 2003). Select EGFL repeats of tenascin cytotactin, an extracellular matrix protein, bind with ultra-low affinity and a fast off-rate, leading to compartmentalization of active receptors at the cell surface without internalization of either receptor or ligand (Iyer et al., 2007). This direct activation of EGFR is distinct from the indirect activation that occurs secondary to integrin-mediated signaling elicited by other domains of these proteins ( Jones et al., 1997). This may be characteristic of binding of EGFL repeats in general (Schenk and Quaranta, 2003; Tran et al., 2004, 2005), allowing for a novel signaling mechanism distinct from classical growth factors. We hypothesized that compartmentalization of liganded EGFR at the cell surface would lead to selective activation of intracellular cascades and that this would influence the overall cell response. Specifically, we postulated that in response to the transient nature of binding of EGFL repeats, EGFR would activate PLCγ and m-calpain at the cell surface, leading to enhanced migration but lacking the tonic intracellular activation of ERK that drives the cells toward proliferation. Our results indicate relatively robust activation of molecules associated with the migratory cascade downstream of EGFR in response to Ten14, leading to preferential activation of cell migration at concentrations of Ten14 that failed to stimulate proliferation. This work presents a novel mechanism by which ECM proteins containing EGFL repeats signal EGFR, leading to a more selective and directed cell response from a potentially pleiotropic receptor.