Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin–Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.

信号分子组装成微米大小的簇是由多价蛋白质-蛋白质相互作用驱动的，例如在去氧肾上腺素-Nck（Nck1或Nck2）复合物中发现的相互作用。去氧肾上腺素跨膜受体尾部多个酪氨酸残基的磷酸化会诱导细胞质接头蛋白 Nck 的募集，该蛋白通过其三重 SH3 结构域与各种效应子结合，从而导致肌动蛋白组装。去氧肾上腺素聚集的生理后果尚不清楚。在这里，我们证明去氧肾上腺素磷酸化调节足细胞膜簇的形成。我们还揭示了聚类和内吞作用之间的联系，这似乎是由去氧肾上腺素酪氨酸磷酸化和 Nck SH3 结构域信号传导的阈值水平驱动的。最后，我们揭示了足细胞损伤期间去氧肾上腺素酪氨酸磷酸化、去氧肾上腺素定位和肾小球滤过屏障完整性的短暂变化之间的体内相关性。总而言之，我们的结果表明去氧肾上腺素磷酸化决定簇内效应蛋白的组成，以动态调节去氧肾上腺素周转和足细胞健康。