Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells.
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 7x10(5) 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-alpha). 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.
Perez-Torres
Marianela
M
Department of Biochemistry, University of Puerto Rico-Medical Sciences Campus, PO Box 365067 San Juan, Puerto Rico.
Valle
Blanca L
BL
Maihle
Nita J
NJ
Negron-Vega
Lisandra
L
Nieves-Alicea
Rene
R
Cora
Elsa M
EM
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism
D010455:Peptides; D011480:Protease Inhibitors; D020033:Protein Isoforms; D016211:Transforming Growth Factor alpha; C076715:pervanadate; D014638:Vanadates; D004815:Epidermal Growth Factor; C010024:4-aminophenylmercuriacetate; D066246:ErbB Receptors; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D010662:Phenylmercuric Acetate