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 B-12, is a novel type of high molecular weight receptor consisting of a 27 CUB ((c) under bar omplement components C1r/C1s, (U) under bar egf, and (b) under bar one morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence, In addition to binding the vitamin B-12-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 (Lys(74)-Glu(109)) 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 B-12 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.