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 epidermal growth factor receptor (EGFR) expression using different immunological reagents. Previous studies have used EGFR-directed antibodies that cannot distinguish between full-length and 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 coexpressed 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 710-735) is 35% identical in sequence with a 31-mer carboxy-terminal sEGFR peptide and exhibits an approximately 5-fold lower affinity for anti-sEGFR than the homologous 31-mer sEGFR peptide does. 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 three-dimensional 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 integrins in complex processes such as cell adhesion and survival signaling.

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 is indicated by the hatched box. A peptide matching the first 31 amino acids (residues 604–634) of the unique C-terminus of sEGFR (underlined) was used to generate anti-sEGFR as described by Christensen et al. (10).

FIGURE 2

Expression of EGFR, sEGFR and p140 in carcinoma-derived cell lines is detected by immunoblot analysis with anti-sEGFR. Panel A. An anti-sEGFR reactive band of 90-kDa, which is also reactive with a monoclonal antibody against EGFR extracellular domain (not shown) is detected in the lysates of CHO cells stably expressing sEGFR (CHO-sEGFR), as well as in A2780, ES-2, and OVCAR-8 cells (closed arrowhead). A second immunoreactive band of 140-kDa is detected in JEG-3, HEY, A2780, A2780-CP-R, ES-2, and IGROV-1 cells (open arrowhead). Panel B. An anti-EGFR (intracellular domain specific) reactive band of 170 kDa is detected in the lysates of CHO cells stably expressing EGFR (CHO-EGFR), as well as in JEG-3, ES-2, HEY, IGROV-1, OVCAR-5 cells.

FIGURE 3

Chromatographic purification steps for p140 as determined by immunoblot analysis with anti-sEGFR. Panel A. Depletion of p140 from JEG-3 lysates with SP Sepharose at each indicated pH. After incubation of SP Sepharose at the indicated pH, supernatants were assayed for unbound p140 by immunoblot analysis with anti-sEGFR. SP Sepharose efficiently bound p140 in buffers at pH ≤ 5.0. Panel B. Elution of p140 from SP Sepharose equilibrated with 50 mM formic acid (pH 4.0) using 0 to 1 M NaCl gradient. After incubation of SP Sepharose with cell lysate (pH 4.0) with the indicated NaCl solution, supernatants were assayed for unbound p140 by immunoblot analysis with anti-sEGFR. p140 was efficiently eluted from SP Sepharose at pH 4.0 by ≥ 400 mM NaCl. Panel C. Elution of p140 from a hydroxylapatite column equilibrated with 20 mM sodium phosphate buffer (pH 6.8) using a gradient of 20 to 185 mM sodium phosphate buffer. Eluted fractions were assayed for the presence of p140 by immunoblot analysis with anti-sEGFR. p140 was eluted from hydroxylapatite with 90–110 mM sodium phosphate, pH 6.8.

FIGURE 4

Expression of BASP-1, α5-integrin, and β1-intergin in JEG-3 and HEY cell lysates as detected by immunoblot analysis. JEG-3 (panel A) and HEY (panel B) cell lysates were resolved by SDS-PAGE as a single band spanning the width of the gel, and transferred to PVDF membrane. The membrane was immunoblotted using a multichannel Immentics blotter, with antibodies directed against BASP-1, α5-integrin, β1-integrin, sEGFR, and EGFR (cytoplasmic domain) at the indicated dilutions. Solid arrowheads indicate migration of BASP-1, α5-integrin, β1-integrin, and EGFR, respectively. Open arrowheads indicate migration of p140. Of the tested species, only a band reactive with anti-α5-integrin co-migrated with p140.

FIGURE 5

Anti-sEGFR recognizes α5-integrin. Panel A. Immunoblot of p140 immunoprecipitated from JEG-3 cell lysates with anti-α5-integrin antibody and reacted with anti-sEGFR (right panel) or secondary antibody alone (left panel). Solid arrowhead indicates mobility of p140. Open arrowhead indicates mobility of a minor ~95 kDa band, which we postulate is a product of α5-integrin. Panel B. Immunoblot of recombinant α5/β1-integrin reacted with different dilutions of anti-sEGFR, as indicated, confirming that anti-sEGFR recognizes α5-integrin.

FIGURE 6

Expression of α5-integrin is detected by immunoblot analysis with anti-sEGFR and anti-α5-integrin antibodies. α5-integrin deficient CHO B2 cells express very low endogenous α5-integrin as detected by anti-sEGFR (Panel A) or anti-α5-integrin (Panel B). Lysates of CHO B2 cells stably expressing human α5-integrin (CHO-α5) contain species reactive as a band by reducing SDS-PAGE followed by anti-sEGFR immunoblot, or a higher molecular mass band by non-reducing SDS-PAGE followed by anti-α5-integrin immunoblot (following manufacturer’s instructions for this antibody). CHO-sEGFR and JEG-3 cells are included as negative and positive controls for p140/α5-integrin, respectively. Note different exposure lengths for CHO-sEGFR and JEG-3 vs. CHO B2 and CHO-α5 lanes in Panel A for clarity.

FIGURE 7

Panel A. Diagram summary of α5-integrin amino acid sequence. Predicted sites of CNBr cleavage are shown with arrowheads. Peptides recovered by LC ESI MS/MS from a anti-sEGFR 31-mer immunoreactive 27-kDa gel band generated by CNBr hydrolysis are underlined: ⁶⁹⁵VTAPPEAEYSGLVR⁷⁰⁸ and ⁷³⁹AGASLWGGLR⁷⁴⁸. Potential sites of N-linked glycosylation are highlighted in bold. Potential sites of cyanogen bromide cleavage are indicated with solid arrowheads. Panel B. Sequence comparison of α5-integrin residues 588–737 with the sEGFR 31-mer peptide. Regions of α5-integrin that have >35% identity with sEGFR were aligned according to SIM analysis (http://www.expasy.org/tools/sim-prot.html). Amino acids sharing sequence identity are highlighted with an asterisk.

FIGURE 8

Immunoblots of recombinant α5/β1 integrin digested by CNBr hydrolysis. Panel A. Time-course of CNBr hydrolysis of recombinant α5/β1 integrin. CNBr hydrolysis was terminated at the times indicated. A precursor-product relationship is evident resulting in an anti-sEGFR-reactive band of ~27-kDa. Panel B. CNBr hydrolysis of deglycosylated α5-integrin. Prior to CNBr hydrolysis, recombinant α5/β1-integrin was treated with PNGaseF. An anti-sEGFR-reactive protein of ~17-kDa was generated by this procedure; this peptide corresponds to α5-integrin peptide 588–737 (see Figure 7), indicating that the anti-sEGFR epitope of α5-integrin is located between amino acids 588–737.

FIGURE 9

Surface plasmon resonance sensorgrams of anti-sEGFR binding to a sEGFR vs. two α5-integrin peptides is indicated. Absorbance in relative units (RU) is plotted vs. time (sec) for immobilized anti-sEGFR in the presence of a sEGFR 31-mer peptide 604–634 (panel A), a α5-integrin peptide 637–649 (panel B), and a α5-integrin peptide 710–735 (panel C). Graphs of RU vs. peptide concentration are included as insets.

FIGURE 10

(A) Anti-sEGFR promotes compaction of suspended CHO B2 cells. Compaction of CHO-P3 and CHO-α5 was compared by suspending cells in hanging drops in the presence of PBS (panels A, B), rabbit IgG (panels C, D) or anti-sEGFR (panels E, F). In general, anti-sEGFR appears to markedly enhance aggregate compaction. This effect is greater for α5-integrin expressing cells than for control cell lines. (B) Quantification of aggregate size. Image analysis was used to calculate average aggregate size for parental and α5-integrin expressing cells incubated in hanging drop cultures in PBS, rabbit IgG, and anti-sEGFR. Statistical analysis by ANOVA revealed a significant difference in aggregate size in response to anti-sEGFR treatment (p<0.0001). Tukey’s Multiple Comparisons Test revealed a pair-wise difference between the following groups of cells: CHO-P3-anti-sEGFR and CHO- α5-anti-sEGFR (p<0.001); CHO-α5-IgG and CHO-α5-anti-sEGFR (p<0.01); CHO-P3-IgG and CHO-P3-anti-sEGFR (p<0.001). As expected, no difference in size was detected between CHO-P3 and CHO- P3-IgG (p>0.05) or CHO-α5 and CHO-α5 IgG (p>0.05).