Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells

Marianela Perez-Torres, Blanca L. Valle, Nita Jane Maihle, Lisandra Negron-Vega, Rene Nieves-Alicea, Elsa M. Cora

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2907-2918
Number of pages12
JournalExperimental Cell Research
Volume314
Issue number16
DOIs
StatePublished - Oct 1 2008

Fingerprint

Epidermal Growth Factor Receptor
Protein Isoforms
Metalloproteases
Conditioned Culture Medium
Cell Line
Amino Acid Sequence
Exosomes
Serine Proteinase Inhibitors
Tandem Mass Spectrometry
Keratinocytes
Epidermal Growth Factor
Digestion
Adenocarcinoma
Acetates
Hot Temperature
High Pressure Liquid Chromatography
Phosphorylation
Breast Neoplasms
Ligands
Carcinoma

Keywords

  • EGFR
  • Ectodomain shedding
  • Metalloproteases
  • Soluble receptors

ASJC Scopus subject areas

  • Cell Biology

Cite this

Perez-Torres, M., Valle, B. L., Maihle, N. J., Negron-Vega, L., Nieves-Alicea, R., & Cora, E. M. (2008). Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells. Experimental Cell Research, 314(16), 2907-2918. https://doi.org/10.1016/j.yexcr.2008.07.013

Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells. / Perez-Torres, Marianela; Valle, Blanca L.; Maihle, Nita Jane; Negron-Vega, Lisandra; Nieves-Alicea, Rene; Cora, Elsa M.

In: Experimental Cell Research, Vol. 314, No. 16, 01.10.2008, p. 2907-2918.

Research output: Contribution to journalArticle

Perez-Torres, M, Valle, BL, Maihle, NJ, Negron-Vega, L, Nieves-Alicea, R & Cora, EM 2008, 'Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells', Experimental Cell Research, vol. 314, no. 16, pp. 2907-2918. https://doi.org/10.1016/j.yexcr.2008.07.013
Perez-Torres, Marianela ; Valle, Blanca L. ; Maihle, Nita Jane ; Negron-Vega, Lisandra ; Nieves-Alicea, Rene ; Cora, Elsa M. / Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells. In: Experimental Cell Research. 2008 ; Vol. 314, No. 16. pp. 2907-2918.
@article{91ed1f9f3ab04312b7095b24ef282cb0,
title = "Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells",
abstract = "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.",
keywords = "EGFR, Ectodomain shedding, Metalloproteases, Soluble receptors",
author = "Marianela Perez-Torres and Valle, {Blanca L.} and Maihle, {Nita Jane} and Lisandra Negron-Vega and Rene Nieves-Alicea and Cora, {Elsa M.}",
year = "2008",
month = "10",
day = "1",
doi = "10.1016/j.yexcr.2008.07.013",
language = "English (US)",
volume = "314",
pages = "2907--2918",
journal = "Experimental Cell Research",
issn = "0014-4827",
publisher = "Academic Press Inc.",
number = "16",

}

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 Jane

AU - Negron-Vega, Lisandra

AU - Nieves-Alicea, Rene

AU - Cora, Elsa M.

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

UR - http://www.scopus.com/inward/record.url?scp=51749089748&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=51749089748&partnerID=8YFLogxK

U2 - 10.1016/j.yexcr.2008.07.013

DO - 10.1016/j.yexcr.2008.07.013

M3 - Article

C2 - 18687326

AN - SCOPUS:51749089748

VL - 314

SP - 2907

EP - 2918

JO - Experimental Cell Research

JF - Experimental Cell Research

SN - 0014-4827

IS - 16

ER -