Characterization of an epithelial similar to 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B-12 and binds receptor-associated protein

By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B-12 (IF-B-12)-binding renal epithelial protein of similar to 460 kDa was copurified together with the transcobalamin-B-12-binding 600-kDa receptor, megalin, IF-B-12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same similar to 460-kDa protein. Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B-12 to a site distinct from the RAP binding site. The high affinity binding of IF-B-12 was dependent on complex formation with vitamin B-12. Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-Co-57-B-12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B-12 to lysosomes. Polyclonal antibodies recognizing the similar to 460-kDa receptor inhibited the uptake, Immunohistochemistry of kidney and intestine showed colocalization of the IF-B-12 receptor and megalin in both tissues. In conclusion, we have identified the epithelial IF-B-12-binding receptor as a similar to 460-kDa RAP-binding protein facilitating endocytosis.