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 characterize d in renal proximal tubule and yolk sac epithelia. We utilized a yolk sac teratocarcinoma cell line, BN/MSV, which expresses two glycoprotei ns, megalin/gp330 and gp280, also detected in renal proximal tubule an d yolk sac epithelial cells. In this study, we further define the loca lization, internalization and intracellular trafficking of both protei ns in BN/MSV cells. For this purpose, double indirect immunofluorescen ce and immunoelectron microscopy were performed on BN/MSV cells. In ad dition, antibodies against gp280 and gp330, coupled to colloidal gold particles, were used as tracers to follow the endocytosis and recyclin g of the two glycoproteins in BN/MSV cells. BSA and MOPC21 (a nonspeci fic monoclonal antibody) coupled to gold particles were used as contro ls. We have previously shown that gp280 and megalin/gp330 were localiz ed in clathrin-coated pits; both proteins can also be detected in nonc oated areas. Vesicular labeling has previously been seen in the cytopl asm of permeabilized BN/MSV cells. The results of the present study re vealed that the glycoproteins were colocalized in the same cells. Ultr astructural analysis of ultracryosections of BN/MSV cells revealed a l ocalization of both proteins in coated invaginations and small and lar ge endocytic vacuoles. In addition, gp280 and megalin/gp330 were found in the Golgi apparatus and in the granular endoplasmic reticulum. Fur thermore, 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 smal l and large endocytic vacuoles. While anti-gp330 is predominantly loca lized in large endocytic vacuoles, the anti-gp280 gold is mainly conce ntrated in the tubulovesicular structures, which probably correspond t o dense tubules known to enable membrane recycling in the epithelial c ells of renal proximal tubules. Moreover, anti-gp330 gold particles ar e also found in lysosomes, but to a lesser extent than BSA and MOPC21 gold particles, which were highly concentrated in lysosomes. In conclu sion, 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.