The intrinsic factor-vitamin B12 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 interactions. An antiserum raised against the purified protein labeled the apical cell surface of the epithelial cells in proximal tubules and the luminal surface of small intestine. Ten peptide sequences, derived from the protein, all matched the recently published sequences for rat (Moestrup, S. K., Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H., Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I., Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for intrinsic factor-vitamin B12 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 supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its subunits obtained by analytical ultracentrifugation. In a search for a potential assembly domain, we identified a region of heptad repeats in the N-terminal part of the cubilin sequence. Computer-assisted analysis 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-helix.
Lindblom
A
A
Department of Internal Medicine, Malmö General Hospital, Lund University, S-214 01 Malmö, Sweden.
Quadt
N
N
Marsh
T
T
Aeschlimann
D
D
Mörgelin
M
M
Mann
K
K
Maurer
P
P
Paulsson
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006801:Humans; D007150:Immunohistochemistry; D007437:Intrinsic Factor / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D014805:Vitamin B 12 / Q000378:metabolism*
D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C029486:intrinsic factor-cobalamin receptor; C036746:intrinsic factor-vitamin B 12 complex; D007437:Intrinsic Factor; D014805:Vitamin B 12