Identification and characterization of two distinct ligand binding regions of cubilin.
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.
Yammani
R R
RR
Division of Gastroenterology and Hepatology, Department of Medicine, Medical College of Wisconsin and the Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53226, USA.
Seetharam
S
S
Seetharam
B
B
D000418:Albumins / Q000378:metabolism; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002474:Cell-Free System; D004220:Disulfides; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D008024:Ligands; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012709:Serum Albumin / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014805:Vitamin B 12 / Q000737:chemistry
D000418:Albumins; D004220:Disulfides; D008024:Ligands; D011956:Receptors, Cell Surface; D012709:Serum Albumin; C029486:intrinsic factor-cobalamin receptor; D004815:Epidermal Growth Factor; D014805:Vitamin B 12; D002118:Calcium