Department of Obstetrics, Gynecology, and Reproductive Science, Yale School of Medicine, 310 Cedar Street, New Haven, CT 06520-8063, USA.
Biochemistry. 2011 May 24;50(20):4309-21. doi: 10.1021/bi200126j. Epub 2011 Apr 27.
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.
表皮生长因子受体（EGFR/HER1/ERBB1）编码的蛋白质被作为许多人类癌症的诊断、预后和治疗生物标志物进行研究。这些组织/肿瘤生物标志物的临床应用受到限制，部分原因是使用不同免疫试剂观察到的表皮生长因子受体（EGFR）表达存在不一致的结果。以前的研究使用了不能区分全长和可溶性 EGFR（sEGFR）表达的 EGFR 定向抗体。我们生成并鉴定了一种针对位于 sEGFR 独特的 78 个氨基酸羧基末端序列内的 31 个氨基酸肽（残基 604-634）的抗 sEGFR 多克隆抗血清。在这里，我们使用该抗体证明 sEGFR 与多种癌源性细胞系中的 EGFR 共表达。此外，我们还表明，该抗体还检测到另一种约 140 kDa 的蛋白质（p140）。严格的生化特征鉴定表明，该第二蛋白为α5 整联蛋白。我们表明，α5 整联蛋白的小牛结构域中的 26 个氨基酸肽（残基 710-735）在序列上与 31 个氨基酸羧基末端 sEGFR 肽有 35%的同源性，并且对 sEGFR 的亲和力比对同源 31 个氨基酸 sEGFR 肽低约 5 倍。我们得出结论，sEGFR 的羧基末端和α5 整联蛋白的小牛 1 结构域共享一个序列同源区域，导致它们与抗 sEGFR 相互免疫反应。我们还证明抗 sEGFR 促进体外三维组织凝聚力和紧实度，这进一步表明 sEGFR 和α5 整联蛋白之间存在功能联系，以及小牛 1 结构域在细胞黏附中的作用。这些结果对 EGFR 和 sEGFR 作为癌症生物标志物的研究具有重要意义，也为细胞表面 EGFR 同工型与整联蛋白之间相互作用的机制提供了新的见解，在细胞黏附和存活信号等复杂过程中。
