Removal of the Membrane-anchoring Domain of Epidermal Growth Factor Leads to Intracrine Signaling and Disruption of Mammary Epithelial Cell Organization

Abstract

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti–EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti–EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.

Induced Autocrine Signaling through the Epidermal Growth Factor Receptor Contributes to the Response of Mammary Epithelial Cells to Tumor Necrosis Factor α

“…Cell Culture and Reagents-Human mammary epithelial cells (HMECs), strain 184A1 (22), were routinely cultured in DFCI-1 medium supplemented with 12.5 ng/ml human epidermal growth factor (Calbiochem) as previously described (20). Batimastat was generously provided by British Biotech Pharmaceuticals Ltd. Recombinant soluble human TNF, the EGFR kinase inhibitor PD153035, and MMP-3 Inhibitor I were purchased from Calbiochem.…”

“…Batimastat was generously provided by British Biotech Pharmaceuticals Ltd. Recombinant soluble human TNF, the EGFR kinase inhibitor PD153035, and MMP-3 Inhibitor I were purchased from Calbiochem. The EGFR-neutralizing monoclonal antibody 225 (225 mAb) was purified from hybridoma supernatants as previously described (20). All other reagents were of cell culture or molecular biology grade.…”

“…The induction of MMP-9 is likely to be physiologically important, because it has been shown that, in the case of rat MECs, peptide inhibitors of MMP-9 disrupt TNF-stimulated growth and three-dimensional organization on reconstituted basement membrane (46). In HMECs, inhibition of EGFR autocrine signaling by 225 mAb also disrupts cellular organization on a reconstituted extracellular matrix (20). Because both MMP-9 expression and EGFR signaling are commonly increased in mammary tumors, it will be important in future studies to investigate whether the expression of the proteins is coordinately regulated in cancer and whether inflammatory mediators like TNF are involved.…”

Cbl-dependent Ubiquitination Is Required for Progression of EGF Receptors into Clathrin-coated Pits

“…In nontransfected cells ( Figure 2B), the EGF-EGFR complex was efficiently endocytosed upon incubation with 60 ng/ml EGF at 37°C for 10 min, and labeling for EGF was found in multivesicular endosomes. It should be noted that such high concentrations of EGF induce both clathrin-dependent and -independent endocytosis as EGFinduced endocytosis of EGFR through clathrin-coated pits is saturable (Lund et al, 1990;Wiley et al, 1998). In cells overexpressing d.n.…”

“…Overexpressing H-Ras17N significantly reduced phosphorylation of MAPK upon addition of EGF ( Figure 3B), demonstrating that the expression of H-Ras17N affected intracellular signaling. Because EGF-induced endocytosis of EGFR through clathrin-coated pits is saturable (Lund et al, 1990;Wiley et al, 1998), we additionally performed an internalization assay using a low concentration of 125 I-EGF (1 ng/ml) in order to quantify the effect of H-Ras17N on clathrin-dependent endocytosis of the EGFR. As demonstrated (Figure 3C), overexpression of H-Ras17N did not inhibit the clathrin-dependent endocytosis of 125 I-EGF.…”