Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability.
Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor-cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55-60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85-90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.
Ahuja
Rajiv
R
Division of Gastroenterology and Hepatology, Departments of Medicine, Medical College of Wisconsin and Zablocki Veterans Administration Medical Center, Milwaukee, WI 53295, USA.
Yammani
Raghunatha
R
Bauer
Joseph A
JA
Kalra
Seema
S
Seetharam
Shakuntla
S
Seetharam
Bellur
B
D000818:Animals; D007668:Kidney / Q000378:metabolism; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*
C527306:Amn protein, rat; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D011506:Proteins; D011956:Receptors, Cell Surface; C029486:intrinsic factor-cobalamin receptor