Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK.
Traffic. 2018 Jan;19(1):44-57. doi: 10.1111/tra.12531. Epub 2017 Nov 6.
Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans-endocytosis of the inter-cellular receptor-ligand EphB-ephrinB complex is required. The molecular mechanism underlying trans-endocytosis is poorly defined. Here we show that the process is clathrin- and Eps15R-mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co-cultures of EphB2- and ephrinB1-expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2-mediated cell repulsion as shown in a rescue experiment in the EphB2 co-culture assay where wild type Eps15R but not the clathrin-binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans-endocytosis and thereby cell repulsion.
Eph 受体及其配体 ephrins 的表达在成年和胚胎组织的边界形成和形态发生中具有重要作用。EphB 受体和 ephrinB 配体是跨膜蛋白，在不同的细胞中表达，它们的相互作用驱动细胞排斥。为了发生细胞排斥，需要细胞间受体-配体 EphB-ephrinB 复合物的转胞吞作用。转胞吞作用的分子机制尚未明确。在这里，我们使用稳定表达 EphB2 和 ephrinB1 的 Co115 结肠癌细胞系表明，该过程是网格蛋白和 Eps15R 介导的。在 EphB2 和 ephrinB1 表达细胞的共培养物中，Eps15R 的敲低显著降低了细胞排斥，而 Eps15 的敲低则没有。Eps15R 中的一个新的相互作用基序 DPFxxLDPF 被证明可以在体外直接结合网格蛋白末端结构域。此外，Eps15R 与网格蛋白的相互作用是 EphB2 介导的细胞排斥所必需的，这在 EphB2 共培养测定中的挽救实验中得到了证明，野生型 Eps15R 而不是网格蛋白结合突变体挽救了细胞排斥。这些结果首次提供了证据表明，Eps15R 与网格蛋白一起控制 EphB/ephrinB 的转胞吞作用，从而控制细胞排斥。
