TY - JOUR
T1 - Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells
AU - Perez-Torres, Marianela
AU - Valle, Blanca L.
AU - Maihle, Nita J.
AU - Negron-Vega, Lisandra
AU - Nieves-Alicea, Rene
AU - Cora, Elsa M.
N1 - Funding Information:
The authors thank Dr. Jill Reiter (Yale School of Medicine, New Haven, CT) for her helpful suggestions and advice, Trace Christensen (Mayo Clinic, Rochester, MN) for his skilled technical support and Ti Badri for her editing support. The work described in this report was funded by the NIH/NCI (CA73859 and CA85133), the UPR-MSC MBRS-RISE Program (R25GM61838), UPR-MSC MBRS-SCORE Program (S06GM08225), NIH CA 096297 and NIH (COBRE P20RR016439).
PY - 2008/10/1
Y1 - 2008/10/1
N2 - Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7 × 105 receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-α). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.
AB - Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7 × 105 receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-α). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.
KW - EGFR
KW - Ectodomain shedding
KW - Metalloproteases
KW - Soluble receptors
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U2 - 10.1016/j.yexcr.2008.07.013
DO - 10.1016/j.yexcr.2008.07.013
M3 - Article
C2 - 18687326
AN - SCOPUS:51749089748
SN - 0014-4827
VL - 314
SP - 2907
EP - 2918
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 16
ER -