Internalization and recycling of glycoprotein 280 in BN/MSV yolk sac epithelial cells: a model system of relevance to receptor-mediated endocytosis in the renal proximal tubule.

The processes of endocytosis and recycling have been well characterized in renal proximal tubule and yolk sac epithelia. We utilized a yolk sac teratocarcinoma cell line, BN/MSV, which expresses two glycoproteins, megalin/gp330 and gp280, also detected in renal proximal tubule and yolk sac epithelial cells. In this study, we further define the localization, internalization and intracellular trafficking of both proteins in BN/MSV cells. For this purpose, double indirect immunofluorescence and immunoelectron microscopy were performed on BN/MSV cells. In addition, antibodies against gp280 and gp330, coupled to colloidal gold particles, were used as tracers to follow the endocytosis and recycling of the two glycoproteins in BN/MSV cells. BSA and MOPC21 (a nonspecific monoclonal antibody) coupled to gold particles were used as controls. We have previously shown that gp280 and megalin/gp330 were localized in clathrin-coated pits; both proteins can also be detected in noncoated areas. Vesicular labeling has previously been seen in the cytoplasm of permeabilized BN/ MSV cells. The results of the present study revealed that the glycoproteins were colocalized in the same cells. Ultrastructural analysis of ultracryosections of BN/MSV cells revealed a localization of both proteins in coated invaginations and small and large endocytic vacuoles. In addition, gp280 and megalin/gp330 were found in the Golgi apparatus and in the granular endoplasmic reticulum. Furthermore, incubation of BN/MSV cells in the presence of colloidal gold particles labeled with antibodies to gp280 and gp330 demonstrated an internalization from the apical membrane through coated pits into small and large endocytic vacuoles. While anti-gp330 is predominantly localized in large endocytic vacuoles, the anti-gp280 gold is mainly concentrated in the tubulovesicular structures, which probably correspond to dense tubules known to enable membrane recycling in the epithelial cells of renal proximal tubules. Moreover, anti-gp330 gold particles are also found in lysosomes, but to a lesser extent than BSA and MOPC21 gold particles, which were highly concentrated in lysosomes. In conclusion, our results show that gp280 is internalized in BN/MSV cells and that anti-gp280 gold is accumulated in a recycling compartment. Thus, we suggest that gp280 is a receptor for endocytosis.