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.

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...

The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B 12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.

Cubilin is a 460-kDa protein functioning as an endocytic receptor for intrinsic factor vitamin B 12 complex in the intestine and as a receptor for apolipoprotein A1 and albumin reabsorption in the kidney proximal tubules and the yolk sac. In the present study, we report the identification of cubilin as a novel transferrin (Tf) receptor involved in catabolism of Tf. Consistent with a cubilinmediated endocytosis of Tf in the kidney, lysosomes of human, dog, and mouse renal proximal tubules strongly accumulate Tf, whereas no Tf is detectable in the endocytic apparatus of the renal tubule epithelium of dogs with deficient surface expression of cubilin. As a consequence, these dogs excrete increased amounts of Tf in the urine. Mice with deficient synthesis of megalin, the putative coreceptor colocalizing with cubilin, also excrete high amounts of Tf and fail to internalize Tf in their proximal tubules. However, in contrast to the dogs with the defective cubilin expression, the megalin-deficient mice accumulate Tf on the luminal cubilin-expressing surface of the proximal tubule epithelium. This observation indicates that megalin deficiency causes failure in internalization of the cubilin-ligand complex. The megalin-dependent, cubilin-mediated endocytosis of Tf and the potential of the receptors thereby to facilitate iron uptake were further confirmed by analyzing the uptake of 125 I-and 59 Fe-labeled Tf in cultured yolk sac cells.

Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism.

Prompted by reports that MT-I interferes with renal uptake of the megalin ligand ␤2-microglobulin in conscious rats, we tested the hypothesis that megalin binds MT and mediates its uptake. Three lines of evidence suggest that binding of MT to megalin is critical in renal proximal tubular uptake of MT-bound heavy metals. First, MT binds megalin, but not cubilin, in direct surface plasmon resonance studies. Binding of MT occurs at a single site with a Kd ϳ10 Ϫ4 and, as with other megalin ligands, depends on divalent cations. Second, antisera and various known megalin ligands inhibit the uptake of fluorescently labeled MT in model cell systems. Anti-megalin antisera, but not control sera, displace Ͼ90% bound MT from rat renal brush-border membranes. Megalin ligands including ␤2-microglobulin and also recombinant MT fragments compete for uptake by megalin-expressing rat yolk sac BN-16 cells. Third, megalin and fluorescently labeled MT colocalize in BN-16 cells, as shown by fluorescent microscopic techniques. Follow-up surface plasmon resonance and flow cytometry studies using overlapping MT peptides and recombinant MT fragments identify the hinge SCKKSCC region of MT as a critical site for megalin binding. These findings suggest that disruption of the SCKKSCC motif can inhibit proximal tubular MT uptake and thereby eliminate much of the renal accumulation and toxicity of heavy metals such as cadmium, gold, copper, and cisplatinum. cadmium; cisplatin; cubilin; proximal tubules HEAVY METALS COMPLEXED TO metallothionein (MT) class I disturb many functions within the proximal tubules, but the entry route of these complexes into epithelial cells remains unknown (10,14,16,23,24,27,31,44). The best-studied heavy metal at present is cadmium. Environmental and occupational exposure to cadmium are widespread but mostly chronic and low level (1). Whether ingested or inhaled, the majority of absorbed cadmium eventually complexes with MT (9, 32), which is produced by several tissues and is largely intracellular but readily detectable at low levels in the circulation. The resulting heavy metal complex Cd-MT, containing seven cadmium ions (Fig. 1), is small enough (ϳ7 kDa) to be freely filtered through the renal glomerulus into the proximal tubular fluid, before reuptake into proximal tubular cells (15).Although neither the apoprotein nor the zinc complex appears toxic, Cd-MT is a renal tubular toxin whose damage is marked by proteinuria, glucosuria, and aminoaciduria, or in more severe cases, acute tubular necrosis or chronic renal failure (37). Conflicting reports implicate different transporters or, more likely, receptor-mediated pathways in the cellular uptake of Cd-MT (4,5,15,22,28,47). At least some of the uncertainty concerning uptake pathways arises from the use of in vivo and in vitro models that differ significantly in their behavior. For example, while CdCl 2 is more toxic than Cd-MT to cultured rat kidney proximal tubules and LLC-PK 1 cells, Cd-MT shows greater in vivo nephrotoxic effects (26,27,35). Furthermore, in vi...

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The molecular mechanisms controlling proximal tubule reabsorption of proteins have been much elucidated in recent years. Megalin and cubilin constitute two important endocytic receptor proteins involved in this process. Although structurally very different the two receptor proteins interact to mediate the reabsorption of a large number of filtered proteins, including carrier proteins important for transport and cellular uptake of several vitamins, lipids and other nutrients. Dysfunction of either protein results in tubular proteinuria and is associated with specific changes in vitamin metabolism due to the defective proximal tubular reabsorption of carrier proteins. Additional focus on the two receptors is attracted by the possible pathogenic role of excessive tubular protein uptake during conditions of increased filtration of proteins, and by recent findings implicating members of the low density lipoprotein-receptor family, which includes megalin, in the transduction of signals by association with cytoplasmic proteins.