Epidermal growth factor receptor (EGFR) dysregulation is observed in many human cancers and is both a cause of oncogenesis and a target for chemotherapy. We previously showed that partial charge neutralization of the juxtamembrane (JX) region of EGFR via the EGFR R1–6 mutant construct induces constitutive receptor activation and transformation of NIH 3T3 cells, both from the plasma membrane and from the ER when combined with the ER-retaining L417H mutation (Bryant et al. in J Biol Chem 288:34930–34942, 2013). Here, we use chemical crosslinking and immunoblotting to show that these mutant constructs form constitutive, phosphorylated dimers in both the plasma membrane and the ER. Furthermore, we combine this electrostatic perturbation with conformationally-restricted receptor mutants to provide evidence that activation of EGFR R1–6 dimers requires functional coupling both between the EGFR extracellular dimerization arms and between intracellular tyrosine kinase domains. These findings provide evidence that the electrostatic charge of the JX region normally serves as a negative regulator of functional dimerization of EGFR.

在许多人类癌症中观察到表皮生长因子受体 (EGFR) 失调，它既是肿瘤发生的原因，也是化疗的靶点。我们之前表明，通过 EGFR R1-6 突变构建体对 EGFR 的近膜 (JX) 区域的部分电荷中和可诱导 NIH 3T3 细胞的组成型受体激活和转化，当与 ER-保留 L417H 突变（Bryant et al. in J Biol Chem 288:34930–34942, 2013）。在这里，我们使用化学交联和免疫印迹来表明这些突变体构建体在质膜和内质网中形成组成型磷酸化二聚体。此外，我们将这种静电扰动与构象受限的受体突变体相结合，以提供证据表明 EGFR R1-6 二聚体的激活需要 EGFR 细胞外二聚化臂之间和细胞内酪氨酸激酶结构域之间的功能耦合。这些发现提供了证据，表明 JX 区域的静电荷通常作为 EGFR 功能二聚化的负调节因子。