Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Abstract

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 1251-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The Vitamin B12 and other cobalamins (B12)ti form complexes with three mammalian tissue fluid proteins. These include gastric intrinsic factor, transcobalamin (TC) present in plasma, and haptocorrin found in most secretions. Intrinsic factor secreted from the stomach is essential for the intestinal uptake of vitamin B12, whereas TC facilitates the cellular uptake of B12 from plasma and various tissue fluids into a wide spectrum of cells (1, 2). Lack of synthesis of either intrinsic factor (3) or TC (4) leads to hematological, gastrointestinal, and/or neurological disorders.B12 is widely distributed in the mammalian organism and high concentrations are seen in the liver and kidney. Animal studies (5-7) have shown that the kidney plays a major role in the regulation of the plasma B12 level by maintaining a pool of free B12. On the other hand, liver B12 is largely bound to the Bi2-dependent enzymes, methylmalonyl coenzyme A mutase and methionine synthetase. During deficiency, B12 is released from the kidney rather than from the enzyme-bound pool of the vitamin in the liver and other organs (7). Radiolabeled B12 taken up in the kidney is localized exclusively in the renal proximal tubules (8). B12 is only taken up in the kidney when it is bound to TC, and if administrated B12 exceeds the saturation level of plasma TC, free B12 is excreted in the urine (9). In agreement with this fact, high-affinity binding sites for complexes, but not for free B12, have been shown on the luminal surface of isolated rat (10) and hog tubule cells (11). Interestingly, the proportional uptake in the kidney of dietary B12 or intravenously administrated TC-B12 is higher at a high B12 load in the organism, and a regulatory mechanism for uptake and storage of B12 in the kidney has been suggested (5,6,8,10,12).Megalin [previously designated glycoprotein 330 (gp330) or Heymann Nephritis autoantigen] is a 600-kDa endocytosismediating membrane protein expressed in the tubule epithelium (13-15) and some other absorptive epithelia, e.g., in yolk sac, lung, retina, and inner ear (16,17). Megalin belongs to the low density lipoprotein receptor gene family (18) and has been shown to bind different substances including clusterin (19), polybasic aminoglycoside drugs (20), and several of the proteins binding to the homologous 600-kDa a2-macroglobulin receptor/low density lipoprotein receptor-related protein (a2MR/LRP)-e.g., the 39-44 kDa receptor-associated protein (RAP) and plasminogen activators (free or in complex with type-1 inhibitor) (for reviews, see refs. 21 and 22

Collagenase-3 Binds to a Specific Receptor and Requires the Low Density Lipoprotein Receptor-related Protein for Internalization

“…The receptors have high affinity for their ligands and broad specificity. They recognize not only lipoproteins but also a variety of nonlipoprotein ligands, including urokinase and tissue plasminogen activator with their inhibitors and participate in different physiological processes (26,(37)(38)(39)(40)(41)(42)(43)(44)(45). Ten members of this family are known to date: the LDL receptor itself, ␣ 2 -macroglobulin receptor/low density lipoprotein receptor-related protein (␣ 2 MR/LRP), very low density lipoprotein receptor, Heymann nephritis antigen/ megalin/gp330, chicken vitellogenin receptor, Drosophila Yolkless, chicken LR8B, placental calcium sensor protein (29), the newly discovered apolipoprotein E receptor 2 (apoER2) (30), and LR11 (31).…”

Functional and Structural Characterization of Synthetic HIV-1 Vpr That Transduces Cells, Localizes to the Nucleus, and Induces G2 Cell Cycle Arrest

“…In parallel, cells were also treated with a 100-fold excess of unlabeled sVpr or Vpu . Cells were incubated for specified times at 37°C, and the distribution of intra-and extracellular radioactivity was determined as described (30). Briefly, medium was removed, and the cell layer was washed with PBS and lysed with 1% Triton X-100 in PBS.…”

Evidence for megalin-mediated proximal tubular uptake of L-FABP, a carrier of potentially nephrotoxic molecules

“…26 The majority of the ligands that are internalized by megalin are transported via endocytotic vesicles to lysosomes, resulting in their dissociation and degradation. 38 Megalin also regulates the endocytosis of complexes of vitamins and carrier proteins such as transcobalamin, 39 vitamin D-binding protein (DBP), 40 and retinol-binding protein, 41 indicating the importance of megalin in both maintaining vitamin homeostasis and metabolizing certain vitamins, notably the renal hydroxylation of vitamin D. 42 The megalin-mediated uptake of 25-(OH) vitamin D 3 by PTC is followed by lysosomal degradation of DBP and subsequent conversion of 25-(OH) vitamin D 3 to 1,25-(OH) 2 vitamin D 3 , which is then returned to the circulation. 40 Megalin, as well as its cooperative receptor cubilin, is also involved in the PTC uptake of Clara cell secretory protein that is translocated from the airway lumen to the circulation and serves as a carrier of steroid hormones and polychlorinated biphenyl metabolites.…”