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, cubilin-deficient 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.

Steroid hormones are central regulators of a variety of biological processes. According to the free hormone hypothesis, steroids enter target cells by passive diffusion. However, recently we demonstrated that 25(OH) vitamin D ₃ complexed to its plasma carrier, the vitamin D-binding protein, enters renal proximal tubules by receptor-mediated endocytosis. Knockout mice lacking the endocytic receptor megalin lose 25(OH) vitamin D ₃ in the urine and develop bone disease. Here, we report that cubilin, a membrane-associated protein colocalizing with megalin, facilitates the endocytic process by sequestering steroid–carrier complexes on the cellular surface before megalin-mediated internalization of the cubilin-bound ligand. Dogs with an inherited disorder affecting cubilin biosynthesis exhibit abnormal vitamin D metabolism. Similarly, human patients with mutations causing cubilin dysfunction exhibit urinary excretion of 25(OH) vitamin D ₃ . This observation identifies spontaneous mutations in an endocytic receptor pathway affecting cellular uptake and metabolism of a steroid hormone.

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 cubilin-mediated 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.

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 B12 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.