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

The EGFR/HER/ErbB family of receptor tyrosine kinases consists of EGFR/HER1, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4. These receptors signal via both ligand-dependent (1) and -independent (2) pathways to regulate cell growth, differentiation, survival, migration, and adhesion, as well as oncogenic transformation. Consequently, it is not surprising that ErbB/HER overexpression and/or mutational activation play major roles in the etiology and progression of human cancer. In support of this concept, a number of HER receptor-targeted therapeutics have been FDA-approved (e.g., cetuximab, gefitinib, trastuzumab) for the treatment of adult solid malignancies, while many others are under clinical investigation (3). EGFR is the prototypic member of the ErbB/HER receptor family; it is a ~170-kDa glycoprotein consisting of an amino-terminal extracellular domain (ECD), a short transmembrane domain, and a carboxy-terminal intracellular domain (Figure 1). The ECD is divided into four subdomains, I–IV; subdomains I and III regulate ligand binding, while subdomains II and IV regulate receptor homo- or hetero-dimerization. In addition to full-length EGFR, we have reported the isolation of a cDNA clone encoding a soluble EGFR isoform that contains most of the ECD, but which lacks the transmembrane and intracellular domains. This EGFR isoform, sEGFR, arises from an alternate splice event, and incorporates a short region of novel coding sequence, including a termination codon and two polyadenylation sequences (4). Specifically, sEGFR contains sequences encompassing EGFR ECD subdomains I–III, most of subdomain IV, and the unique 78 amino acid carboxy-terminus (Figure 1) (5). This alternatively spliced sEGFR protein product is co-expressed with EGFR in diverse human tissues and also is the precursor for the major circulating isoform of EGFR in human blood, as discussed below. FIGURE 1 Schematic diagram illustrating the structure of EGFR compared to sEGFR. The extracellular domain is divided into four subdomains (I–IV); the transmembrane domain is indicated by the black box; the alternate C-terminal amino acid sequence of sEGFR ... While sEGFR does not contain a canonical transmembrane domain, it is, nevertheless, associated with the cell surface (6), and we recently have shown that cell surface sEGFR is released into the extracellular milieu via a complex process that is regulated by the extracellular matrix protein fibronectin (Wilken et al., unpublished experiments). Purification of anti-EGFR reactive protein(s) from human blood reveals one major protein species which contains peptides derived from the unique carboxy-terminus of sEGFR as determined by mass spectrometry (7). Taken together, these observations support the hypothesis that sEGFR released from the cell surface enters the bloodstream via the lymphatic system and thoracic duct. Translational studies have shown that low serum sEGFR concentrations are associated with the presence of ovarian cancer (7), poor overall survival in endometrial cancer patients (8), and responsiveness to aromatase inhibitor therapy in breast cancer patients (9). To develop further the clinical utility of sEGFR as a serum and tumor biomarker we have generated polyclonal antisera directed against a 31-mer peptide (residues 604–634) located within the unique carboxy-terminal sequence of sEGFR (Figure 1) (10). This affinity-purified antibody (anti-sEGFR) is specific for sEGFR, and does not detect the full-length EGFR, or any other HER receptor family member (10). Here, we report the further characterization of this anti-sEGFR antibody, including an apparent evolutionary convergence between the unique sEGFR carboxy-terminal sequence and a short bioactive sequence within the extracellular region of α5-integrin, i.e., the calf-1 domain, which coordinates ligand-dependent associations between the α and β subunits of the integrin heterodimer (11).