No abstract

Steroids destined for intracellular metabolic conversion or binding to nuclear receptors are believed to cross cell membranes by passive diffusion. According to this free hormone hypothesis, steroids bound to plasma carrier proteins are inactive because they cannot reach their intracellular targets (1). However, recent data show that carrier proteins may greatly facilitate steroid uptake by endocytosis of steroid-carrier complexes followed by intracellular release of the steroid (2, 3). Megalin, a member of the low density lipoprotein receptor family abundant in kidney proximal tubules, mediates endocytic uptake of complexes between the steroid 25 ( Megalin binds a large number of structurally unrelated ligands, and coreceptors may confer ligand specificity by sequestering and presenting their cargo to megalin (4). For example, intrinsic factorvitamin B 12 complex ) is taken up in the intestine by a tandem receptor-mediated mechanism; the complex is first bound to a receptor, cubilin, anchored to the outer leaflet of the plasma membrane possibly by an amphipathic helix (5), followed by endocytosis of cubilin and its cargo mediated by megalin (6, 7). The pivotal role of intestinal cubilin is underscored by the vitamin B 12 deficiency observed in patients with Imerslund-Gräsbeck disease characterized by defective cubilin incapable of binding IF-B 12 (8). These patients have low molecular weight proteinuria in addition to megaloblastic anemia, indicating dysfunction of cubilin coexpressed with megalin in kidney proximal tubules. However, whereas the role of cubilin in the intestine is well characterized, the physiological role in the kidney remains elusive.Here, we identify cubilin as an important coreceptor in the endocytic pathway for retrieval of 25(OH)D 3 -DBP complexes by megalin-mediated endocytosis in the kidney. We show that absence of cubilin or inhibition of its function markedly reduces cellular uptake of the steroid-carrier complex, and animals or patients lacking functional cubilin are characterized by abnormal vitamin D metabolism. This study identifies patients with mutations in an endocytic pathway that regulates steroid hormone metabolism. Materials and MethodsLigands, Receptors, and Antibodies. DBP was purified from human serum (2). Receptor-associated protein (RAP) was produced in Escherichia coli (9); 3 H-25(OH)D 3 was from Amersham Pharmacia, and 25(OH)D 3 was from Dr. A.-M. Kissmeyer (Leo Pharmaceutical Products, Ballerup, Denmark). Biotin-25(OH)D 3 was synthesized by coupling 25(OH)D 3 -3-(3Ј-aminopropyl)ether (10) with aminocaproic acid-biotin-4-nitrophenyl ester (Pierce) (11). Sterol-DBP complexes were prepared by incubating DBP with 10 to 100-fold excess labeled or unlabeled 25(OH)D 3 (2). Uncomplexed steroid was removed by gel filtration or dialysis. Human retinolbinding protein (RBP) was from Dr. G. Alexander (University of Oslo, Norway). Rabbit megalin and cubilin were purified as reported (5).The primary antibodies used were rabbit anti-human DBP and anti-human RBP (Dako), goat anti-h...

Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of cubilin, indicating a mutual dependency of cubilin and amnionless to form a functional membrane receptor complex. The cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, cubilindeficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds cubilin and megalin. In addition, we observed cubilin-independent reabsorption of the "specific" cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.