The intrinsic factor vitamin B-12 receptor, cubilin, is assembled into trimers via a coiled-coil alpha-helix

A large protein was purified from bovine kidney, using selective extraction with EDTA to solubilize proteins anchored by divalent cation-dependent int eractions. An antiserum raised against the purified protein labeled the api cal cell surface of the epithelial cells in proximal tubules and the lumina l surface of small intestine. Ten peptide sequences, derived from the prote in, all matched the recently published sequences for rat (Moestrup, S. K. K ozyraki, R., Kristiansen, M., Kaysen, J. H., Helm Rasmussen, H., Brault, D. , Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verro ust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a recep tor for intrinsic factor-vitamin B-12 complexes, identifying the protein as bovine cubilin. In electron microscopy, a three-armed structure was seen, indicating an oligomerization of three identical subunits. This model was s upported by the M-r values of about 1,500,000 for the intact protein and 44 0,000 for its subunits obtained by analytical ultracentrifugation. In a sea rch for a potential assembly domain, we identified a region of heptad repea ts in the N-terminal part of the cubilin sequence. Computer-assisted analys is supported the presence of a coiled-coil alpha-helix between amino acids 103 and 132 of the human cubilin sequence and predicted the formation of a triple coiled-coil. We therefore conclude that cubilin forms a noncovalent trimer of identical subunits connected by an N-terminal coiled-coil alpha-h elix.