PMID:11581259

Citation	Yammani, RR, Seetharam, S and Seetharam, B (2001) Identification and characterization of two distinct ligand binding regions of cubilin. J. Biol. Chem. 276:44777-84
Abstract	Using polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6-8 bound both intrinsic factor-cobalamin (vitamin B(12); Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110-115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca(2+) dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60-65%. However, a combination of CUB domains 7 and 8 or 6-8 was needed to demonstrate significant Ca(2+) dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6-8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds.
Links	PubMed Online version:10.1074/jbc.M106419200
Keywords	Albumins/metabolism; Alkylation; Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Calcium/metabolism; Cell-Free System; Disulfides; Dogs; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor/metabolism; Ligands; Microsomes/metabolism; Molecular Sequence Data; Pancreas/metabolism; Plasmids/metabolism; Polymerase Chain Reaction; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; Rats; Receptors, Cell Surface/chemistry; Receptors, Cell Surface/metabolism; Reverse Transcriptase Polymerase Chain Reaction; Serum Albumin/metabolism; Spectrometry, Fluorescence; Structure-Activity Relationship; Transcription, Genetic; Vitamin B 12/chemistry
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RAT:CUBN	enables	GO:0038023: signaling receptor activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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