Molecular dissection of the intrinsic factor-vitamin B12 receptor, cubilin, discloses regions important for membrane association and ligand binding.
Cubilin, the receptor for intrinsic factor-vitamin B12, is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In addition to binding the vitamin B12-carrier complex, cubilin also binds receptor-associated protein. To delineate the structures for membrane association and ligand binding we established a panel of stable transfected Chinese hamster ovary cells expressing overlapping segments of rat cubilin. Analysis of conditioned media and cell extracts of transfected cells revealed that the N-terminal cubilin region conveys membrane association. Helical plotting of this region demonstrated a conserved amphipathic helix pattern (Lys74-Glu109) as a candidate site for hydrophobic interactions. Ligand affinity chromatography and surface plasmon resonance analysis of the secreted cubilin fragments showed ligand binding in the CUB domain region. Further dissection of binding-active fragments localized the binding site for intrinsic factor-vitamin B12 to CUB domains 5-8 and a receptor-associated protein-binding site to CUB domains 13-14. In conclusion, the N-terminal cubilin region seems crucial for membrane association, whereas the CUB domain cluster harbors distinct sites for ligand binding.
Department of Medical Biochemistry, University of Aarhus, Denmark.
M
M
Kristiansen
R
R
Kozyraki
C
C
Jacobsen
E
E
Nexø
P J
PJ
Verroust
S K
SK
Moestrup
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007437:Intrinsic Factor / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013552:Swine; D014805:Vitamin B 12 / Q000378:metabolism*
D008024:Ligands; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C029486:intrinsic factor-cobalamin receptor; C036746:intrinsic factor-vitamin B 12 complex; D007437:Intrinsic Factor; D014805:Vitamin B 12