Differential Roles of Grb2 and AP‐2 in p38MAPK‐ and EGF‐Induced EGFR Internalization

Abstract

The epidermal growth factor receptor (EGFR) is an important regulator of normal growth and differentiation, and it is involved in the pathogenesis of many cancers. Endocytic downregulation is central in terminating EGFR signaling after ligand stimulation. It has been shown that p38 MAPK activation also can induce EGFR endocytosis. This endocytosis lacks many of the characteristics of ligand-induced EGFR endocytosis. We compared the two types of endocytosis with regard to the requirements for proteins in the internalization machinery. Both types of endocytosis require clathrin, but while epidermal growth factor (EGF)-induced EGFR internalization also required Grb2, p38 MAPK-induced internalization did not. Interestingly, AP-2 knock down blocked p38 MAPK-induced EGFR internalization, but only mildly affected EGF-induced internalization. In line with this, simultaneously mutating two AP-2 interaction sites in EGFR affected p38 MAPK-induced internalization much more than EGF-induced EGFR internalization. Thus, it seems that EGFR in the two situations uses different sets of internalization mechanisms.

Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress

“…S2B). By contrast, as expected (18,29,31), when cells were treated with anisomycin (Aniso) at 37°C or when EGF-treated cells were warmed to 37°C for 15 min (EGF37C), EGFR were effectively internalized ( Fig. 2A).…”

“…For example, binding of the E3 ubiquitin ligase CBL at EGFR pY 1069 (13)(14)(15) or indirectly through the adaptor protein Grb2, which binds primarily at pY 1092 (16), are both involved in EGFR ubiquitinylation and down-regulation (17). Although not an exclusive mechanism, EGFR internalization mainly involves clathrin and the AP-2 clathrin adaptor complex (12, 18 -22) in addition to Grb2 (18,23,24). EGFR internalization and recycling in response to stress-induced p38 MAPK activation requires AP-2, but not Grb2 (18), and is reportedly independent of receptor kinase activity, tyrosine phosphorylation, and ubiquitination (6 -8).…”

“…Although not an exclusive mechanism, EGFR internalization mainly involves clathrin and the AP-2 clathrin adaptor complex (12, 18 -22) in addition to Grb2 (18,23,24). EGFR internalization and recycling in response to stress-induced p38 MAPK activation requires AP-2, but not Grb2 (18), and is reportedly independent of receptor kinase activity, tyrosine phosphorylation, and ubiquitination (6 -8).…”

EGF receptor trafficking: consequences for signaling and cancer

“…The EGFR itself can also become directly activated via modification of cysteine residues located in its active site [93]. This activation is accompanied by receptor internalization and elicits endocytic trafficking/signaling events that have not been fully characterized but are either p38- [85,86] or Src-dependent [94,95], and clathrin- and AP2 adaptor-dependent [96].…”

The AP-2 Clathrin Adaptor Mediates Endocytosis of an Inhibitory Killer Cell Ig-like Receptor in Human NK Cells

“…Clathrin forms a triskelion structure that drives endocytic vesicle formation, but requires adaptors to bind surface receptors. The AP-2 clathrin adaptor is directly implicated in the internalization of many receptors, including transferrin receptor (TfR), LDLR and EGFR (21–23). AP-2 is a heterotetrameric complex composed of α- and β-adaptin that interact with clathrin and the plasma membrane, μ2, which associates with cargo containing tyrosine-based motifs, and σ2, which is involved in binding cargo containing dileucine-based motifs (19, 21).…”