INVITED REVIEW
Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule

Erik Ilsø Christensen and Henrik Birn

Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

The multiligand, endocytic receptors megalin and cubilin are colocalized in the renal proximal tubule. They are heavily expressed in the apical endocytic apparatus. Megalin is a 600-kDa transmembrane protein belonging to the low-density lipoprotein-receptor family. The cytoplasmic tail contains three NPXY motifs that mediate the clustering in coated pits and are possibly involved in signaling functions. Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is a 460-kDa receptor with no transmembrane domain and no known signal for endocytosis. Because the two receptors bind each other with high affinity and colocalize in several tissues, it is highly conceivable that megalin mediates internalization of cubilin and its ligands. Both receptors are important for normal tubular reabsorption of proteins, including albumin. Among the proteins normally filtered in the glomeruli, cubilin has been shown to bind albumin, immunoglobulin light chains, and apolipoprotein A-I. The variety of filtered ligands identified for megalin include vitamin-binding proteins, hormones, enzymes, apolipoprotein H, albumin, and beta 2- and alpha 1-microglobulin. Loss of these proteins and vitamins in the urine of megalin-deficient mice illustrates the physiological importance of this receptor.

proteinuria; vitamin D; vitamin B12; retinol; low-density lipoprotein- receptor family


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

MEGALIN IS A MULTILIGAND, endocytic receptor belonging to the low-density lipoprotein (LDL)-receptor family. It is heavily expressed in the renal proximal tubule. A long list of ligands for megalin has been identified establishing an important role for the tubular uptake of filtered proteins. Among the ligands are vitamin binding proteins and several hormones, suggesting an additional role of megalin in the metabolism and homeostasis of essential vitamins, including vitamin D, as well as calcium.

Cubilin, also known as the intestinal intrinsic factor-cobalamin receptor, is coexpressed with megalin in the renal proximal tubule. Although structurally very different from megalin, it has many similar features, being a multiligand, endocytic receptor sharing several ligands with megalin and being important for the tubular reabsorption of proteins. In addition, megalin has been shown to bind cubilin and is most likely involved in the endocytosis of this receptor.

The present review will focus on megalin and cubilin in the kidney, their structural features, mutual interaction, potential signaling function, and their role in tubular protein reabsorption, vitamin metabolism, as well as calcium homeostasis.


    HISTORY
TOP
ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

Megalin

Megalin was originally identified as the antigen in Heymann nephritis of rats. It was purified from rat kidney brush border and named gp330 on the basis of molecular weight as estimated by its mobility during gel electrophoresis (43). Megalin was localized in the apical endocytic pathway of renal proximal tubule as well as in the glomeruli (43). Later, it was identified in several other epithelia (for a recent review, see Ref. 16). Many proteins were identified as ligands, including receptor-associated protein (RAP), lipoproteins, enzymes, and enzyme inhibitors, suggesting the role of megalin as a scavanger endocytic receptor. In 1994, the protein was cloned by Saito et al. (81), showing it to be a 600-kDa glycoprotein and suggesting the name "megalin." In 1996, megalin-deficient mice was produced by gene targeting by Willnow et al. (92), adding significant new information about the important role of this receptor.

Cubilin

Cubilin was identified as the target of teratogenic antibodies produced by the injection of renal brush-border preparations into rabbits (78). It was named gp280 on the basis of the estimated molecular weight and localized to the apical endocytic apparatus of renal proximal tubule and the visceral epithelia of yolk sac (78). No ligands were identified until 1997, when gp280 was shown to be identical to the intestinal intrinsic factor-cobalamin receptor (84). Also, in 1997 RAP was shown to be a ligand for cubulin, followed by several other proteins. The receptor was cloned by Moestrup et al. (65) in 1998 and shown to be a 460-kDa glycoprotein with no apparent cytoplasmic domain, and the name "cubilin" was suggested on the basis of its structure dominated by complement subcomponents C1r/C1s, Uegf, and bone morphogenic protein-1 (CUB) domains.


    STRUCTURE
TOP
ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

Megalin

Megalin is an ~4,600-amino acid transmembrane protein (Fig. 1) with a large NH2-terminal extracellular domain, a single transmembrane domain, and a short cytoplasmic tail (81). The protein belongs to the LDL-receptor family (76), sharing common features with the following mammalian receptors including the LDL receptor, the LDL-receptor-related protein (LRP), the very-low-density lipoprotein (VLDL) receptor, and the apolipoprotein E (apo E) receptor-2 (reviewed in Ref. 29). Both rat (81) and human (37) megalin has been cloned, and the nonglycosylated molecular mass is estimated to 517 kDa (81). The cytoplasmic tail contains three NPXY motifs mediating the binding to adaptor proteins and the clustering into coated pits. In addition, this motif may serve signaling functions (75). Also, the cytoplasmic domain contains several Src homology 3 and one Src homology 2 recognition sites (37). The extracellular domain contains four cystein-rich, complement-type repeats, probably constituting the ligand binding regions separated by epidermal growth factor (EGF) precursor homology domains containing YWTD repeats responsible for the pH-dependent release of ligands (24). The human megalin gene has been located to chromosome 2q24-q31 (46).


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Fig. 1.   Illustration of the structural organization and interaction between cubilin and megalin. The C1r/C1s, Uegf, and bone morphogenic protein-1 (CUB) domains in cubilin and the complement-type repeats in megalin represent the ligand binding regions of the 2 receptors. Cubilin is a peripheral membrane protein, whereas megalin is a transmembrane protein with 3 cytoplasmic NPXY motifs, directing the receptor into coated pits. A number of filtered ligands for each of the 2 receptors in the kidney proximal tubule are also depicted, in addition to albumin and receptor-associated protein (RAP), which bind to both receptors. Finally, the binding of cubilin to megalin and the megalin-mediated internalization of the ligand-receptor-receptor complex are indicated. Apo, apolipoprotein; RBP, retinol-binding protein; TC, transcobalamin; DBP, vitamin D-binding protein; EGF, epidermal growth factor; PTH, parathyroid hormone.

Cubilin

Cubilin is an ~3,600-amino acid protein with no transmembrane domain (Fig. 1). The complete DNA sequences of rat (65), human (52), and canine (96) cubilin have been identified, showing a nonglycosylated molecular mass of 400 kDa. It has little structural homology with other known endocytic receptors. The extracellular domain contains 27 CUB domains. The CUB domains most likely constitute the ligand binding domains, and the binding site for intrinsic factor-cobalamin has been located within CUB domains 5-8 whereas the binding site for RAP is located within CUB domains 13-14 (54). The CUB domains are preceded by a stretch of 110 amino acids followed by 8 EGF-type repeats. The initial amino acid stretch contains a furin cleavage site, which may indicate proteolytic processing in the trans-Golgi network (52). The NH2-terminal region seems essential to membrane anchoring of the protein. This segment contains an amphipatic helix structure with some similarity to the lipid binding regions of apolipoproteins, which may contribute to the anchoring of the receptor in the membrane (54). The human cubilin gene has been located to chromosome 10p12.33-p13 (52).


    EXPRESSION AND SYNTHESIS
TOP
ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

Megalin

Megalin is expressed in many epithelial cells (49, 99; recently reviewed in Ref. 16), in particular absorptive epithelia facing transcellular fluids, such as the renal proximal tubule, the glomerular podocytes, the choroid plexus, ependymal cells, epididymis, thyroid cells, labyrinthic cells of the inner ear, and the ciliary epithelium of the eye. In addition, megalin is expressed in the visceral yolk sac, type II pneumocytes, the parathyroid hormone (PTH)-secreting cells of the parathyroid gland, the small intestine, the endometrium, the oviduct, and the cytotrophoblast of the placenta. Megalin has also been identified in embryonic tissues such as the trophoectodermic cells and the neuroectoderm (32, 80). During renal development, megalin can be identified in the mesonephros, the nephronic vesicle, and the ureteric bud (80). Megalin is expressed in the S-shaped body later giving rise to both the glomeruli and the proximal as well as the distal tubule. Later during development, megalin is only expressed in the proximal tubules and, to a lesser extent, the glomerulus (80).

In the kidney proximal tubule (Fig. 2), megalin can be localized to the brush border, coated pits, endocytic vesicles (1, 3, 14, 45), and the membrane recycling compartment, dense apical tubules (6, 17, 19). The membrane expression is high in small and large endosomes in the proximal tubule cells but is virtually absent in late endosomes/prelysosomes (16). However, smaller amounts of intact and degraded megalin have been identified in the matrix of the lysosomes (19). In rats the expression of megalin in the proximal tubule brush border varies between different segments, with the highest expression in segment two (19). Megalin has also been identified in the glomerular podocytes of rat kidney (44).


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Fig. 2.   Immunolocalization of megalin (10 nm gold) and cubilin (5 nm gold; arrowheads) in proximal tubule from rat. The 2 receptors are colocalized on the microvilli (MV), in coated pits (CP), apical endosomes (E), and dense apical tubules (arrows) responsible for the apical receptor recycling in these cells. Ultrathin cryosections of rat kidney cortex were incubated with polyclonal sheep anti-rat megalin and rabbit anti-rat cubilin followed by incubation with secondary antibodies coupled to colloidal gold particles. Magnification: ×110,000.

After translation in the rough ER (RER), megalin binds rapidly and with high affinity to the 40-kDa protein RAP. RAP also binds other members of the LDL-receptor family and serves as a chaperone protecting newly synthesized receptor from the early binding of ligands (12, 13, 90, 94). In addition, RAP may be involved in the folding of the receptors (13). RAP deficiency is associated with a significant decrease in the expression as well as a subcellular redistribution of megalin in the proximal tubule (10). A HNEL motif, serving as an RER-retention signal, is present in RAP, and the protein is predominantly located in this organelle (12). Thus RAP is a predominantly intracellular ligand for megalin. However, due to its high-affinity association with megalin, inhibiting the binding of most other ligands, it has served as an important tool for the study of ligand binding to megalin.

Cubilin

Cubilin is highly expressed in the renal proximal tubule and the visceral yolk sac (79), the epithelium of the small intestine (9, 83), the placental cytothrophoblast (33; for a recent review, see Ref. 16), and possibly other tissues including thymus (33), although at present it seems more restricted than the expression of megalin. In the proximal tubule (Fig. 2), cubilin expression very closely resembles that of megalin represented by the brush border and all constituents of the coated pit endocytic and the membrane recycling pathway (78, 84). Cubilin is also identified in lysosomes (84). Similar to megalin, cubilin is expressed in the S-shaped body during renal development whereas later expression is confined to the proximal tubule only (80). So far it has not been identified in the glomerulus.

The posttranslational processing of cubilin may involve furin-mediated cleavage in the trans-Golgi network, as suggested by the finding that affinity-purified human cubilin appears to be truncated at a recognition cleavage site for furin in the NH2-terminal region (52). Furthermore, pulse-chase studies have suggested an unusual processing of cubilin in yolk sac cells, involving the expression of newly synthesized, endoglycosidase H-sensitive cubilin at the plasma membrane although the majority of cubilin is endoglycosidase H resistant (4). This indicates that newly synthesized cubilin is targeted to the plasma membrane and recycled to the Golgi apparatus for final processing, possibly involving both carbohydrate modifications and furin-mediated truncation. Posttranslational processing may be tissue specific, as it was recently shown that ileal cubilin undergoes more extensive NH2-linked glycosylation than renal cubilin (95).

Recently, it was shown that a canine disorder characterized by defective trafficking of cubilin into the apical membranes and a functional cubilin deficiency was not caused by a defect in the cubilin gene. Rather, it was suggested that a yet unknown accessory protein is required for cubilin brush-border expression (96).


    FUNCTION
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ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

Protein Reabsorption

Megalin was recognized early as a endocytic receptor involved in the tubular uptake of proteins. A large number of ligands have been identified (Table 1). Although not all of these can be expected to be present in the glomerular filtrate, many are recognized markers of defective tubular reabsorption, including vitamin D-binding protein (DBP), vitamin A/retinol-binding protein (RBP), beta 2-microglobin, and alpha 1-microglobulin. Analyses in megalin-knockout mice as well as patients with Fanconi syndrome have shown several analogies in urinary protein excretion, suggesting the former to be a model of low-molecular-weight, tubular proteinuria (55). Although fewer ligands have been identified for cubilin (Table 1) so far, recent evidence strongly suggests this protein to be important for tubular protein reabsorption as well. Significant proteinuria is observed in many patients suffering from Imerslund-Gräsbech syndrome (11, 31, 40), a rare vitamin B12-deficiency disease characterized by defective intestinal absorption of the vitamin B12-intrinsic factor complex and recently shown in two Finnish families to be associated with mutations in the cubilin gene (2). Two mutations were identified: a point mutation causing one amino acid substitution in CUB domain 8 affecting the binding of intrinsic factor-B12 (2, 53); and a point mutation expected to activate a cryptic intronic splice site causing an in-frame insertion with several stop codons, predicting a truncation of the receptor in CUB domain 6 (2). Thus variations in the type of mutations causing this disease may explain why some patients have selective intrinsic factor-B12 malabsorption and yet no or only little proteinuria (11, 51). Intense proteinuria is likely to be caused by mutations in the cubilin gene affecting more binding sites or result in the absence of a functional receptor. The importance of cubilin for tubular protein reabsorption is further supported by the proteinuria (27, 28), and in particular albuminuria (8), observed in dogs, characterized by a defective processing and apical insertion of cubilin within the epithelial cells (Fig. 3), causing a vitamin B12 malabsorption syndrome (27, 28).

                              
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Table 1.   Ligands to megalin and cubilin



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Fig. 3.   Immunolocalization of cubilin and endogenous albumin in proximal tubules from the kidney of normal dogs (a and c) and dogs with a defective processing and insertion of cubilin into the apical plasma membrane (b and d). In contrast to normal dogs (a), no cubilin can be identified at the luminal plasma membrane of the affected dogs (b), resulting in a functional cubilin deficiency. As a result, no labeling for albumin is observed in the tubules of the affected dogs (d), reflecting the defective tubular reabsorption of filtered albumin. In normal dogs (c), reabsorbed albumin can be identified in vesicular structures within the proximal tubule cells. Semithin cryosections of dog kidney cortex were incubated with either polyclonal anti-dog cubilin or polyclonal anti-human albumin, followed by peroxidase-labeled secondary antibody and visualization by incubation with diaminobenzidine and H2O2. Magnification: ×500 (a, b, and d); ×1,000 (c).

Thus structural as well as functional defects in either megalin or cubilin are associated with proteinuria, suggesting both receptors to be essential to normal tubular reabsorption of filtered proteins.

Ligands. The known ligands for megalin normally filtered in the glomeruli include DBP, RBP, the vitamin B12/cobalamin plasma carrier protein, transcobalamin (TC)-B12, PTH, insulin, EGF, prolactin, albumin, beta 2- and alpha 1-microglobin, apo H, transthyretin, lysozyme, cytochrome-c, alpha -amylase, and Ca2+ (Table 1). Many of these proteins are either carrier proteins or hormones, suggesting megalin to be involved in the metabolism of vitamins, and in the renal clearance of many filtered hormones by endocytic uptake and degradation. In most cases ligand binding is Ca2+ dependent, and megalin itself binds calcium very strongly (17). It has been shown by site-directed mutagenesis analysis that mutations of basic amino acid residues in aprotinin, a 6-kDa proteinase inhibitor and a ligand for megalin, decrease the affinity for the receptor, suggesting that binding is charge dependent and favored by cationic sites on the ligands (64). However, many ligands are anionic proteins, indicating that it is the distribution of charge rather than the overall isoelectric point that is important for binding, as also suggested previously (20). The binding of almost all ligands can be inhibited by RAP. This high-affinity binding is exploited in both the study of receptor function, ligand binding, as well as for affinity purification of the receptor (66). Megalin has at least two different binding sites for RAP, as demonstrated by surface plasmon resonance analysis. Binding of the first RAP molecule with high affinity was followed by binding of a second molecule with lower affinity (63).

On the basis of immunoprecipitation studies, it was suggested that megalin complexes with the sodium-hydrogen exchanger, NHE-3, in the kidney proximal tubule brush border and that megalin could be involved in the regulation of this transporter by mediating endocytosis (7). Still, further evidence is needed to confirm this hypothesis.

The known ligands for cubilin expected to be present in normal glomerular ultrafiltrate so far only include albumin, immunoglobulin light chains, and apo A-I. Albuminuria is an important marker for renal disease, and a recent study has shown that cubilin is essential to normal renal tubular reabsorption of filtered albumin (8). This, as well as the binding of myeloma light chains (5), may implicate a role for cubilin in the progression of renal disease. Several studies have suggested that tubular uptake of an increased load of filtered proteins, including albumin, contributes to the development of tubular and interstitial inflammation and fibrosis. Thus cubilin-mediated tubular uptake of albumin during states of hyperfiltration of proteins may be an important factor for the development and progression of chronic renal disease. Cubilin also binds RAP (9) although the significance of this is not determined. The binding of apo A-I suggests a role in the renal degradation of this apolipoprotein (34, 51). The kidney is the major site for the catabolism of apo A-I, involving glomerular filtration and subsequent tubular uptake.

Most if not all of the ligands taken up by megalin or cubilin in the proximal tubule are degraded in lysosomes. So far, no evidence for the transcellular transport of protein ligands, including carrier proteins, has been published. However, vitamins, steroids, fatty acids, and other substances carried by proteins, filtered and reabsorbed by the receptors, and released within the cell most likely are transported back to the circulation, possibly after biochemical modifications, as discussed later. Megalin-mediated transcellular transport of proteins has been demonstrated in other tissues such as the thyroid gland for thyroglobulin (60, 61) and in cerebral vascular endothelium and in the choroid epithelium for apo J (101).

Megalin-knockout mice. Much of our recent knowledge on the functions of megalin is based partially on data recovered from the study of megalin-deficient mice. These mice, produced by gene targeting, exhibit severe forebrain abnormalities as well as lung defects (92). Most of them die perinatally; however, approximately 1 of 50 survive to adulthood, constituting a model for the study of megalin function (92). The kidneys of these mice are generally normal; however, ultrastructurally the proximal tubule cells are characterized by a loss of apical endosomes (92), coated pits, and recycling dense apical tubules (21). This probably reflects decreased endocytic activity and supports an important general role for megalin in maintaining proximal tubule endocytosis. The megalin-deficient mice excrete an increased amount of a number of low-molecular-weight plasma proteins in the urine. This is a result of defective tubular reabsorption, as shown by the absence of immunodetectable protein ligands in the proximal tubule cells of deficient mice (8, 18, 36, 55, 67, 71). So far, no significant changes in transport of water, electrolytes, glucose, or amino acids have been described in megalin-deficient mice (55).

Receptor-receptor interactions. As discussed previously, the primary sequence of cubilin does not predict a transmembrane domain (65). Thus cubilin itself does not harbor any obvious sites for interaction with adaptor proteins or other mediators of clathrin-coated endocytosis. However, a high-affinity, Ca2+-dependent, and partially (75%) RAP-inhibitable binding between purified cubilin and megalin has been described (65), suggesting that megalin mediates the cointernalization and possibly recycling of cubilin. The binding between megalin and cubilin appears to be complex. However, by fitting the binding data to a one-binding-site model, a dissociation constant of ~7 nM was measured (65). A similar mechanism involving another member of the LDL-receptor family has been suggested for the internalization of the urokinase receptor-bound-urokinase-inhibitor complex (22). This glycophosphatidylinositol-anchored receptor-ligand complex is internalized by binding to LRP. Recently, it was shown in vitro that the uptake of high-density lipoprotein, which binds to cubilin, was inhibited by anti-megalin antibodies as well as by megalin anti-sense oligonucleotides (33). In addition, treatment with megalin anti-sense oligoneucleotides also reduced the surface expression, but not total expression, of cubilin (33). This may indicate that megalin is also involved in trafficking of cubilin.

In addition to direct receptor interaction, megalin and cubilin seem to share ligands. So far, these include RAP and albumin (Table 1). Thus in the case of albumin both megalin and cubilin are involved in the endocytic uptake (Fig. 1) (8, 98). This may include direct binding of albumin to both receptors as well as receptor-receptor interaction after binding to cubilin.

Vitamin Metabolism and Homeostasis

The megalin-mediated tubular reabsorption of vitamin-carrier proteins appears important for both maintaining vitamin homeostasis and metabolizing certain vitamins, notably the renal hydroxylation of vitamin D. So far, three vitamin-carrier proteins (Table 1), all of which are filtered in the glomeruli, have been identified as ligands for megalin. These are DBP, RBP, and TC-B12. DBP (71) and RBP (18) were both initially identified in the urine of megalin-deficient mice, and TC, which previously was identified as a ligand for megalin (63), has also subsequently been demonstrated in the urine of these mice (Birn H, Willnow T, Nielsen R, Norden AGW, Moestrup S, Nexo E, and Christensen E, unpublished observations).

Filtered plasma vitamin D carrier protein DBP binds to megalin, which mediates the endocytosis of this protein. The megalin-mediated uptake of 25-(OH) vitamin D3 in the proximal tubule is followed by lysosomal degradation of DBP and subsequent conversion of 25-(OH) vitamin D3 to 1,25-(OH)2 vitamin D3, which is then returned to the circulation (71). Considerable amounts of DBP and 25-(OH) vitamin D3 are excreted in the urine of megalin-deficient mice (71). In addition, these mice have reduced plasma vitamin concentration. Furthermore, especially the young mice suffer from severe bone calcification abnormalities, indicating that megalin-mediated tubular uptake is essential for normal calcium homoestasis in these animals (71).

Megalin also mediates the reabsorption of RBP (18) and TC (63) by endocytosis. This must be followed by release of internalized vitamins to the circulation to maintain vitamin homeostasis. Increased amounts of both TC and vitamin B12 can be identified in the urine of megalin-deficient mice in combination with reduced kidney concentrations of B12 (Birn H, Willnow T, Nielsen R, Norden AGW, Moestrup S, Nexo E, and Christensen E, unpublished observations). This shows that megalin is important for preventing urinary loss of the vitamins. In addition, the kidney may serve a vitamin B12 storage function, possibly involving megalin-mediated uptake (Birn H, Willnow T, Nielsen R, Norden AGW, Moestrup S, Nexo E, and Christensen E, unpublished observations). It has been estimated that the tubular vitamin B12 reabsorption is similar to the intestinal uptake (56).

Although the basic molecular mechanisms for the efficient tubular clearance of the carrier proteins appear well established, the subsequent intracellular handling of the vitamins remains to be clarified. After release of vitamins, the carrier proteins most likely are degraded in lysosomes. The vitamins may be transported into the cytoplasm by either diffusion through the vesicular membranes or facilitation by a transport protein. A membrane-associated vitamin B12 transporter has been described (39, 77). Vitamin D3 subsequently undergoes hydroxylation to a more active form, and, similarly, cobalamin may be metabolized into other forms. The final steps, i.e., the release of vitamin and possible coupling to plasma carrier proteins, have not been elucidated. Because vitamin D3 is lipophilic, it has been hypothesized that the vitamin diffuses through the basolateral membranes and meets its carrier protein DBP extracellularly (71). Alternatively, it has been suggested that reabsorbed retinol or B12 is coupled to newly synthesized RBP or TC within the proximal tubule cells and then secreted as a complex (18, 70). These pathways are summarized in Fig. 4.


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Fig. 4.   Schematic illustration of the megalin-mediated uptake of the 3 vitamin carrier protein complexes: DBP-vitamin D3, TC-vitamin B12, and RBP-vitamin A in renal proximal tubule. After megalin-mediated endocytosis via apical coated pits (CP), the complexes accumulate in lysosomes (LYS) for degradation of the proteins, while the receptor is returned to the apical plasma membrane via dense apical tubules (DAT). The intracellular processing of the vitamins may include modifications such as hydroxylation of 25-OH-D3 to 1,25-(OH)2-D3 before basolateral secretion. The mechanisms for secretion of the 3 vitamins remain to be clarified. CV, coated vesicles; SEV, small endocytic vesicles; LEV, large endocytic vesicles; PL, prelysosomes.

The importance of megalin for the uptake and transport of certain vitamins may be indicated by the fact that some of the defects seen in the megalin-deficient mice resemble syndromes of vitamin deficiency. Megalin is located in the visceral epithelium of the yolk sac (15, 25), important for early embryonic nutrition in rodents, and in addition is also in the cytotrophoblast of the placenta (58). Thus megalin may be involved in the transport of vitamins from the maternal circulation to the embryo. It has been hypothesized that megalin and possibly other members of the LDL-receptor family serve an important function mediating tissue-specific uptake of carrier-bound retinoids and steroids, including vitamins and hormones (93). This would provide a mechanism by which certain cell types could accumulate large quantities of steroids beyond what is possible solely by diffusion of free hormones.

Calcium

Megalin serves important functions in calcium homeostasis. As described, megalin mediates proximal tubule endocytosis of the DBP-vitamin D complex, resulting in the renal hydroxylation and activation of vitamin D (71). In addition, megalin mediates tubular uptake and subsequent lysosomal degradation of PTH (36). It has been suggested that this may regulate the amount of PTH available for stimulation of the tubular PTH/PTH-related peptide receptor (36). Megalin is also expressed on the PTH-secreting cells in the parathyroid gland (57) and is, in itself, a very strong calcium binder (17). Thus it may be speculated that the protein serves as a calcium sensor in both the parathyroid gland and the kidney.

Drugs

Megalin has been shown to bind and mediate proximal tubule uptake of several polybasic and potential nephrotoxic substances, including the aminoglycosides gentamicin, netilimicin, and amikacin, as well as polymyxin B (64). These drugs are readily filtered in the glomeruli followed by endocytic uptake and accumulation in the endocytic apparatus and lysosomes of the proximal tubule cells (68, 69, 89). Binding to phospholipids in the apical brush-border membrane has been implicated in the tubular uptake (82); however, the affinity of these drugs for megalin is higher than for phosphatidylinositol, suggesting that aminoglycosides bound to membrane lipids are transferred to megalin for endocytosis (64). Also, the expression of megalin in the labyrinthic cells of the inner ear is intriguing (62, 97) because aminoglycosides as well as polymyxin B are also known to be ototoxic.

Heymann Nehpritis

Megalin was originally identified as the antigen in Heymann nephritis (43), a rat model of human membranous glomerulonephritis (reviewed in Ref. 26). Circulating anti-megalin antibodies bind to megalin expressed in glomerular podocytes, causing destruction of the basement membrane.

So far, no anti-megalin antibodies have been associated with any human renal disease. However, circulating anti-megalin antibodies have been identified in serum from patients with autoimmune thyroiditis as well as some other thyroid diseases (59). Whether these antibodies are involved in the pathogenesis of the underlying autoimmune disease remains to be established.

Signaling Functions?

The megalin cytoplasmic domain contains several regions, suggesting a possible signaling function in addition to its role as endocytic receptor. These include several Src homology 3 and one Src homology 2 recognition sites as well as the NPXY motifs. So far, no definite evidence for the involvement of megalin in signal transduction has been published. However, other members of the LDL-receptor family, the VLDL receptor and apo E receptor-2, have been suggested to be involved in signal transmission initiated by the extracellular matrix protein reelin in the cerebral cortex and cerebellum (88; for recent reviews, see Refs. 35 and 93). This signaling requires the intracellular mammalian disabled protein 1 (Dab1), which was shown to bind to the NPXY motifs on the cytoplasmic tail of both receptors. After binding of Dab1 to the cytoplasmic tail of the VLDL-receptor or apo E receptor-2, Dab1 may be phosphorylated on tyrosine residues, allowing binding and activating nontyrosine kinases. Dab1 has been shown to bind to other members of the LDL-receptor family (38). Also, by using a yeast two-hybrid system, it was recently shown that the cytoplasmic tail of megalin binds the cytosolic disabled protein 2 (Dab2) (72) as well as a number of other cytoplasmic proteins with a potential signaling function (30). Although no specific cellular response has been associated with these interactions involving megalin, Dab2 was identified in rat kidney by Western blotting and was coprecipitated with megalin by using both anti-Dab2 and anti-megalin antibodies (72). This indicates a potential signaling pathway involving megalin in the kidney.

Thus several lines of evidence indicate that members of the LDL-receptor family may be involved in signal transduction although no specific response or overall pathway has been identified for megalin.


    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

Megalin and cubilin constitute two large, endocytic receptors heavily expressed in the endocytic apparatus of the kidney proximal tubule. Absence or dysfunction of either receptor is associated with significant tubular proteinuria, showing that both are important for normal absorption of filtered proteins, including albumin. Although structurally very different, both receptors may be functionally linked. Some ligands are common to both receptors, and megalin-cubilin interaction seems to be important for the endocytosis and recycling of the "peripherally attached" cubilin. Megalin is important for tubular uptake and metabolism of several hormones and vitamin-carrier protein complexes, including the renal activation by hydroxylation of vitamin D. In addition, megalin is involved in the tubular uptake of potential nephrotoxic drugs, including aminoglyocosides. Thus modification of receptor function may be a valuable prospect of future research. This is encouraged by findings suggesting that tubular uptake of an increased load of filtered proteins, including albumin, may contribute to the progression of chronic renal disease. Finally, there is new evidence suggesting that megalin may be involved in signal transduction. No doubt, future studies will help to unfold this potential new aspect of megalin receptor function.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Danish Medical Research Council, the Novo Nordic Foundation, the Danish Biotechnology Program, and University of Aarhus Research Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: E. I. Christensen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: eic{at}ana.au.dk).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
HISTORY
STRUCTURE
EXPRESSION AND SYNTHESIS
FUNCTION
CONCLUSIONS
REFERENCES

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