The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell–cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

受体酪氨酸激酶 (RTK) EphA2 在上皮细胞和内皮细胞中表达，控制细胞与细胞连接的组装。EphA2 还与许多疾病有关，包括癌症。与大多数 RTK 主要以二聚体形式发出信号不同，EphA2 在配体结合后很容易形成高阶寡聚体。在这里，我们研究了 EphA2 信号传导特性与多种配体（包括广泛使用的 ephrinA1-Fc 配体、可溶性单体 m-ephrinA1 和新型工程肽配体）诱导的 EphA2 寡聚体大小之间是否存在相关性。我们使用荧光强度波动 (FIF) 光谱法来表征不同配体诱导的 EphA2 寡聚体群体。有趣的是，我们发现不同的单体和二聚配体诱导EphA2寡聚体的尺寸分布差异很大。我们对 FIF 亮度分布参数和 EphA2 信号参数的比较表明，EphA2 对酪氨酸 588 磷酸化的功效（一种有助于 EphA2 激活的自磷酸化反应）与 EphA2 平均寡聚体大小相关。然而，我们发现其他特征，例如 AKT 抑制的功效和配体偏倚系数，似乎与 EphA2 寡聚体的大小无关。总而言之，这项工作强调了 FIF 在 RTK 信号研究中的实用性，并证明了 EphA2 信号复合物的结构和信号特征之间的定量相关性。