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 i ntrinsic factor-vitamin B-12 (IF-B-12)-binding renal epithelial protei n of similar to 460 kDa was copurified together with the transcobalami n-B-12-binding 600-kDa receptor, megalin, IF-B-12 affinity chromatogra phy of renal cortex membrane from rabbit and man yielded the same simi lar to 460-kDa protein. Binding studies including surface plasmon reso nance analyses of the protein demonstrated a calcium-dependent and hig h 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 fo rmation with vitamin B-12. Light and electron microscope autoradiograp hy of rat renal cortex cryosections incubated directly with IF-Co-57-B -12 and rat proximal tubules microinjected in vivo with the radioligan d demonstrated binding of the ligand to endocytic invaginations of pro ximal tubule membranes followed by endocytosis and targeting of vitami n 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 megali n in both tissues. In conclusion, we have identified the epithelial IF -B-12-binding receptor as a similar to 460-kDa RAP-binding protein fac ilitating endocytosis.