Differential roles of Grb2 and AP-2 in p38 MAPK- and EGF-induced EGFR internalization.

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.

EGF-induced activation of the EGF receptor does not trigger mobilization of caveolae.

Caveolae-dependent endocytosis has recently been proposed in the uptake of EGF receptor (EGFR) at high concentrations of ligand. Consistently, upon incubation of HEp2 and HeLa cells with methyl-beta-cyclodextrin, we observed a small inhibitory effect on endocytosis of ligated EGFR in HEp2 cells. However, immunoelectron microscopy showed the same relative amount of bound EGF localizing to caveolae on incubation with high and low concentrations of EGF, not supporting rapid recruitment of EGFR to caveolae. Live-cell microscopy furthermore demonstrated that incubating HEp2 cells with high concentrations of EGF did not increase the mobility of caveolae. By RNA-interference-mediated knockdown of clathrin heavy chain in HEp2 and HeLa cells, we found that endocytosis of EGFR was efficiently inhibited both at high and low concentrations of EGF. Our results show that caveolae are not involved in endocytosis of EGF-bound EGFR to any significant degree and that high concentrations of EGF do not further mobilize caveolae.

Herceptin-induced inhibition of ErbB2 signaling involves reduced phosphorylation of Akt but not endocytic down-regulation of ErbB2.

The anti-proliferative effect of the ErbB2 specific antibody Herceptin in cells overexpressing ErbB2 has previously been explained by endocytic downregulation of ErbB2. However, in the following, we demonstrate that Herceptin inhibited proliferation of ErbB2 overexpressing cells without downregulating ErbB2. Herceptin did also not induce endocytosis of ErbB2. Herceptin was found to blunt proliferation of SKBr3 cells overexpressing EGFR, ErbB2, and ErbB3 and expressing functional PTEN, probably by recruiting PTEN to the plasma membrane. Akt was found to be constitutively phosphorylated both in SKBr3 cells overexpressing EGFR, ErbB2 and ErbB3, and in SKOv3 cells, overexpressing EGFR and ErbB2. However, phosphorylation of Akt was inhibited by Herceptin only in SKBr3 cells. SKOv3 cells, which lack the tumour suppressor protein Ras homolog member I, was found to have constitutively phosphorylated mitogen activated protein kinase and functionally increased Ras activity. SKOv3 cells further had low expression levels of PTEN. We thus confirm that the anti-proliferative effect of Herceptin in SKBr3 cells is due to recruitment of PTEN to the plasma membrane and conclude that Herceptin does not blunt phosphatidyl inositol 3 kinase-induced growth in cells with constitutive Ras activity. We further conclude that endocytic downregulation of ErbB2 does not contribute to Herceptin's antiproliferative effect.

CD7 expression by CD34+ cells in CML patients, of prognostic significance?

The purpose of the study was to identify a unique immunophenotype of normal or Philadelphia chromosome positive (Ph+) CD34+ cells that might be used to purify normal CD34+ cells from chronic myelogenous leukemia (CML) patients. An immunophenotypical study of CD34+ bone marrow cells of 20 patients with CML at diagnosis and during hydroxyurea treatment, and 39 controls were performed. All patients were Ph+, two patients had variant translocations and three patients displayed cytogenetic signs of clonal evolution. The immature progenitor cell compartment (CD34+ HLA-DR- and CD34+ CD38- cells) was comparable. The CD34+ AC133+ progenitor cell compartment was decreased in CML patients. We found no difference for any of the adhesion molecules examined except for CD62L, where the percentage of CD34+ CD62L+ cells was decreased in CML patients. The number of myeloid progenitors (CD34+ CD33+) was increased at the expense of B-lymphoid progenitors (CD34+ CD10+ and CD34+ CD19+) in CML patients indicating that B-lymphopoiesis is inhibited in CML. The megakaryocytic (CD34+ CD61+) and erythroid (CD34+ CD71+) progenitors were increased in CML patients. The number of CD34+ CD7+ cells was also significantly increased (mean 25.3% vs. 4.9%). However, the level of CD7 expression was quite heterogeneous, and the patients could be separated into two populations according to CD7 expression (more or less than 20% CD7+ CD34+ cells). The Sokal and Hasford risk scores did not differ between CD34+ CD7- CML and CD34+ CD7+ CML, but all patients with signs of disease progression clustered in the CD34+ CD7+ population indicating that the level of CD7 expression on CD34+ cells may be of prognostic importance in CML.

Cholesterol is important in control of EGF receptor kinase activity but EGF receptors are not concentrated in caveolae.

We have investigated the localization and function of the epidermal growth factor receptor (EGFR) in normal cells, in cholesterol-depleted cells and in cholesterol enriched cells. Using immunoelectron microscopy we find that the EGFR is randomly distributed at the plasma membrane and not enriched in caveolae. Binding of EGF at 4 degrees C does not change the localization of EGFR, and by immunoelectron microscopy we find that only small amounts of bound EGF localize to caveolae. However, upon patching of lipid rafts, we find that a significant amount of the EGFR is localized within rafts. Depletion of the plasma membrane cholesterol causes increased binding of EGF, increased dimerization of the EGFR, and hyperphosphorylation of the EGFR. Addition of cholesterol was found to reduce EGF binding and reduce EGF-induced EGFR activation. Our results suggest that the plasma membrane cholesterol content directly controls EGFR activation.

Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies.

EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.