Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D ₃

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...

25-Hydroxyvitamin D3 is a natural chemopreventive agent against carcinogen induced precancerous lesions in mouse mammary gland organ culture

“…It should also be noted that in serum in vivo, as much as 99% of 25(OH)D 3 is bound to the vitamin D binding protein (DBP), and cellular uptake of this complex in the kidney via receptormediated endocytosis is essential for vitamin D homeostasis in vivo [40,41]. Vitamin D is transduced by two separate routes of uptake; active uptake in the presence of DBP and passive uptake in the absence of DBP.…”

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Cubiline : rôle physiopathologique et relations avec la mégaline

“…L'internalisation de ces complexes fait intervenir au moins un co-récepteur, la mégaline. Cette conclusion repose sur l'interaction démontrée entre cubiline et mégaline et la co-localisation étroite des deux récepteurs [6], l'effet inhibiteur des anticorps anti-mégaline sur l'internalisation de la plupart des ligands, et l'absence d'internalisation des complexes ligand-cubiline chez les souris mégaline -/- [13][14][15][16]. Le rôle physiologique de la cubiline varie en fonction du site d'expression considéré.…”