Megalin-mediated endocytosis of transcobalamin-vitamin-B-12 complexes suggests a role of the receptor in vitamin-B-12 homeostasis

Kidney cortex is a main target for circulating vitamin B-12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit I-125-TC-B-12 and human TC-Co-57-B-12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B-12 but no measurable affinity of the vitamin complex for the homologous alpha(2)-macroglobulin receptor (alpha(2)MR)/low density lipoprotein receptor related protein (LRP). I-125-TC-B-12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of I-125-TC-B-12. The TC-B-12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B-12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B-12 homeostasis and fetal vitamin B-12 supply.