Epidermal growth factor receptor signaling intensity determines intracellular protein interactions, ubiquitination, and internalization

Abstract

Ligand activation of the epidermal growth factor receptor (EGFR) causes the binding of Cbls, which leads to EGFR polyubiquitination and internalization through endophilin complexes that contain the adaptor protein SH3-domain encoding, expressed in tumorigenic astrocytes͞Cbl-interacting protein of 85 kDa͞regulator of ubiquitous kinase (SETA͞CIN85͞Ruk). In cells grown at high density, high levels of SETA interfered in the recruitment of Casitas B-lineage (Cbl) proteins to the EGFR and reduced its polyubiquitination, suggesting that SETA has a regulatory function in the formation of the EGFR-Cbl-endophilin complex and in EGFR down-regulation. In a situation where there is EGFR signaling but no internalization or down-regulation, as is the case with the EGFR with exons 2-7 deleted (⌬EGFR) oncogene, these proteins were absent altogether. By using mAb 806, which recognizes an EGFR-activation state and preferentially immunoprecipitates ⌬EGFR, we show that ⌬EGFR did not interact with Cbls, SETA, or endophilin A1, providing a mechanistic explanation for its lack of internalization. As would be expected by the absence of Cbl proteins in the ⌬EGFR complex, the mutant receptor was also not polyubiquitinated. The intracellular C terminus and tyrosine autophosphorylation pattern of ⌬EGFR are similar to wild-type EGFR, but it signals at a lower intensity as determined by levels of EGFR phosphotyrosine. To test the implication that the lack of interaction with the Cbl-SETA-endophilin complex is because of differences in signal intensity, EGFR-expressing cells were treated with tyrphostin AG1478 EGFR inhibitor. Attenuation of wild-type EGFR signal to levels similar to that found in ⌬EGFR resulted in the dissociation of SETA and Cbl proteins and a concomitant attenuation of receptor internalization.

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

“…In contrast to the present study, Schmidt et al (2003) reported that the EGFRvIII does not interact with either Cbl or Cbl-b. In their investigation, HEK 293 cells were transfected with EGFRvIII and either Cbl or Cbl-b.…”

“…Only a small fraction (approximately 10%) of the EGFRvIII protein is active at any given time compared to the WT EGFR that has been stimulated by EGF (Huang et al, 1997;Fernandes et al, 2001). Thus, it is possible that the interaction between the EGFRvIII and the Cbl proteins was below the level of sensitivity of the immunoprecipitation and immunoblotting procedure used by Schmidt et al (2003).…”

“…Supporting this, a recent study reported that the EGFRvIII does not interact with either Cbl or Cbl-b and is not downregulated (Schmidt et al, 2003). Mutations in the Cbl-binding site of several receptor tyrosine kinases (RTKs) result in transforming forms of these RTKs (Peschard et al, 2001;Mancini et al, 2002;Peschard and Park, 2003).…”

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen‐sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR)

“…Recently it has been shown that EGFR signaling intensity determines the rate of internalization of the receptor. 42 In particular, Schmidt et al 42 showed that if autotransphosphorylation of EGFR is reduced below a threshold, a reduction of internalization and downregulation of the receptor occurs. We show here that EGFR autotransphosphorylation is reduced in PC3-AR cells and therefore it is possible that also internalization is consequently reduced.…”

Analysis of Receptor Tyrosine Kinase Internalization Using Flow Cytometry

“…High cell densities could affect the efficiency of ligand binding to the receptor and hence downstream cell-signaling responses. For example, cell density has been shown to affect the internalization of the epidermal growth factor receptor (EGFR) and the formation of the endocytic Cbl-endophilin complex in HEK 293 cells (14).…”