Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor and α5-Integrin

Abstract

Proteins encoded by the epidermal growth factor receptor (EGFR/HER1/ERBB1) gene are being studied as diagnostic, prognostic, and theragnostic biomarkers for numerous human cancers. The clinical application of these tissue/tumor biomarkers has been limited, in part, by discordant results observed for EGFR expression using different immunological reagents. Previous studies have used EGFR-directed antibodies that cannot distinguish between full-length vs. soluble EGFR (sEGFR) expression. We have generated and characterized an anti-sEGFR polyclonal antiserum directed against a 31-mer peptide (residues 604–634) located within the unique 78 amino acid carboxy-terminal sequence of sEGFR. Here, we use this antibody to demonstrate that sEGFR is co-expressed with EGFR in a number of carcinoma-derived cell lines. In addition, we show that a second protein of ~140-kDa (p140) also is detected by this antibody. Rigorous biochemical characterization identifies this second protein to be α5-integrin. We show that a 26 amino acid peptide in the calf domain of α5-integrin (residues 710–35) shares 35% sequence identity with a 31-mer carboxy-terminal sEGFR peptide, and exhibits approximately 5 times lower affinity for anti-sEGFR than does the homologous 31-mer sEGFR peptide. We conclude that the carboxy-terminus of sEGFR and the calf-1 domain of α5-integrin share a region of sequence identity, which results in their mutual immunological reactivity with anti-sEGFR. We also demonstrate that anti-sEGFR promotes 3D tissue cohesion and compaction in vitro, further suggesting a functional link between sEGFR and α5-integrin and a role of the calf-1 domain in cell adhesion. These results have implications for the study of both EGFR and sEGFR as cancer biomarkers, and also provide new insight into the mechanisms of interaction between cell surface EGFR isoforms and integrin in complex processes such as cell adhesion and survival signaling.

Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes

“…ITGA2b 877–956 contains a 36-residue stretch with 28% sequence identity to the EGFR antigen (Supplemental Information Figure S4), which may explain the observed cross reactivity of the EGFR pAb. It was recently reported that anti-sera raised toward the carboxy-terminus of soluble EGFR were cross-reactive with a member of the integrin family (ITGAV)22. While the cross-reactive sequences reported there are different than what we observed, that study highlights the close biological connection between cell-surface growth-factor receptors and integrins.…”

EGFR Soluble Isoforms and Their Transcripts Are Expressed in Meningiomas

“…In addition to whole EGFR, tumor cells express sEGFR proteins that can be generated by alternative mRNA splicing events [13] , [14] , [15] , [16] , [18] , via proteolytic cleavage of the receptor [11] , [12] , [13] , [20] or by aberrant translocation events [21] . In our series, we found that meningiomas expressed the alternatively spliced mRNA transcripts v2, v3 and v4.…”

“…In addition to the full-lenght transmembrane forms, soluble EGFR (sEGFR) isoforms, that comprised solely the ECD portions of the receptor, have been detected in normal and malignant cells, in tissues, and in biological fluids [11] , [12] . These sEGFR proteins can be either generated by alternative mRNA splicing events or via proteolytic cleavage of the receptor [13] , [14] . EGFR gene alternative splicing leads to four transcripts: EGFR variants 1, 2, 3 and 4 (v1, v2, v3 and v4, respectively) mRNA that encode 170-kDa whole receptor and 60-kDa [15] , 80-kDa [16] , [17] and 110-kDa [18] sEGFR isoforms, respectively.…”

EGFR/HER-Targeted Therapeutics In Ovarian Cancer

“…Proteolytic processing of HER2 and HER4 can also give rise to both soluble and truncated membrane-bound HER receptor isoforms [21]. Soluble HER isoforms impart another level of complexity to the regulation of HER signaling, and sEGFR/sHER expression has been shown to influence tyrosine kinase activity, cell cohesion/compaction, cell survival, proliferation, differentiation and migration [21–23]. …”