Proximity Labeling Reveals How Lrp2 Interacts with the Endocytic Machine

LRP2 (Megalin or low-density lipoprotein-related receptor 2), together with Cubilin and Amnionless, is responsible for binding and internalizing a wide range of nutrients and toxins from the kidney's glomerular filtrate by endocytosis. Accordingly, Lrp2 deletion or mutation results in the loss of these ligands into the urine. Yet Lrp2 is essential not only for receptor-mediated but also for fluid-phase endocytosis, implicating a broader role beyond ligand binding. To identify the linkage between Lrp2 and endocytosis, we engineered Lrp2-APEX2-expressing mice and performed biotinylation in vivo to label Lrp2's cytoplasmic partners. We demonstrated the specificity and sensitivity of this technique by mass spectrometric identification of biotinylated proteins from kidney lysate and immunostaining kidney sections. We identified critical endocytic regulators interacting with Lrp2, but also many proteins functionally associated with endocytosis that are not already known to interact with Lrp2. These data suggest that Lrp2 plays a central role in organizing apical membranes through PDZ domain proteins and engages with regulators and molecular motors during endocytosis. These interactions are abolished in the absence of Lrp2.