Abstract
BackgroundEndocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood.Methodology/Principal FindingsWe investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas.Conclusions/SignificanceOur results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis.
Highlights
Signaling via the ubiquitously expressed epidermal growth factor receptor (EGFR) is involved in the regulation of cell motility, proliferation, survival and differentiation [1,2,3]
We demonstrate that the SH3-domain of p130Cas interacts with the proline-rich domain (PRD) of dynamin, and this interaction is essential for p130Cas-mediated inhibition of dynamin phosphorylation and EGFR internalization
It is well known that p130Cas is required for cell adhesioninduced EGFR phosphorylation [20], but it was not known whether p130Cas contributes to cell adhesion-induced increases in EGFR localization at the cell surface
Summary
Signaling via the ubiquitously expressed epidermal growth factor receptor (EGFR) is involved in the regulation of cell motility, proliferation, survival and differentiation [1,2,3]. The autophosphorylation sites in the activated EGFR act as nucleation sites for the formation of specific receptor-signaling protein complexes and downstream kinase (e.g., Erk, Akt) cascades that initiate cell context-specific signaling pathways [6,7]. Endosome-associated EGFRs may continue to interact with compartment-specific signaling proteins. Endocytosis likely plays multiple roles in the regulation of signaling via EGFRs. Endocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation.
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