A Two-receptor Pathway for Catabolism of Clara Cell Secretory Protein in the Kidney*

Regina BurmeisterDagger §, Inger-Margrethe Bøe§, Anders Nykjaer||, Christian Jacobsen||, Soeren K. Moestrup||, Pierre Verroust**, Erik I. ChristensenDagger Dagger , Johan Lund§§, and Thomas E. WillnowDagger ¶¶

From the Dagger  Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin, Germany,  Department of Anatomy and Cell Biology, University of Bergen, 5009 Bergen, Norway, Departments of || Medical Biochemistry and Dagger Dagger  Cell Biology, University of Aarhus, 8000 Aarhus, Denmark, ** Institut National de la Santé et de la Recherche Médicale, Unité 538, 75012 Paris, France, and §§ Department of Molecular Science, AstraZeneca, 22187 Lund, Sweden

Received for publication, November 27, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Clara cell secretory protein (CCSP) is a transport protein for lipophilic substances in bronchio-alveolar fluid, plasma, and uterine secretion. It acts as a carrier for steroid hormones and polychlorinated biphenyl metabolites. Previously, the existence of receptors for uptake of CCSP·ligand complexes into the renal proximal tubules had been suggested. Using surface plasmon resonance analysis, we demonstrate that CCSP binds to cubilin, a peripheral membrane protein on the surface of proximal tubular cells. Binding to cubilin results in uptake and lysosomal degradation of CCSP in cultured cells. Surprisingly, internalization of CCSP is blocked not only by cubilin antagonists but also by antibodies directed against megalin, an endocytic receptor that does not bind CCSP but associates with cubilin. Consistent with a role of both receptors in renal uptake of CCSP in vivo, patients deficient for cubilin or mice lacking megalin exhibit a defect in tubular uptake of the protein and excrete CCSP into the urine. These findings identify a cellular pathway consisting of a CCSP-binding protein (cubilin) and an endocytic coreceptor (megalin) responsible for tissue-specific uptake of CCSP and associated ligands.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Clara cell secretory protein (CCSP)1 is a small homodimeric protein consisting of two 8-kDa subunits linked by disulfide bridges (reviewed in Ref. 1). The protein is predominantly produced in Clara cells, a non-ciliated cell type in the bronchiolar epithelium of the lung, and is secreted into the airway lumen. It represents one of the most abundant soluble proteins in the alveolar fluid (2-4). Some of the CCSP crosses over the lung basal lamina into the plasma from where it is filtered in the glomeruli. Experimental evidence suggests that this filtered CCSP is taken up specifically by epithelial cells of the proximal tubules in the kidney (5). Another site of CCSP expression is the rabbit uterus during the preimplantation phase of pregnancy. In fact, CCSP was first identified as a protein component of rabbit uterine secretion capable of inducing blastulation. It is therefore also known as uteroglobin or blastokinin (6, 7).

CCSP represents an important clinical marker of the health status of the lung and the kidney. In the lung, CCSP expression is induced during cellular differentiation (8, 9). Impaired development of the fetal lung or conditions that harm the adult tissue (e.g. smoking, xenobiotics, or cancer) result in significantly reduced levels of CCSP expression (10-12). In the kidney, uptake of CCSP is dependent on the functional integrity of the proximal tubules, and impaired tubular activity causes urinary excretion of the protein (13).

So far, the physiological significance of CCSP remains unclear. Most notably, CCSP is recognized for its role as a carrier of methylsulfonyl polychlorinated biphenyl metabolites (MeSO2-PCB) in the organism. PCBs are industrial chemicals that, despite being banned from production for many years, remain common in the human population. They accumulate in the lung, in the kidney, and in fatty tissues causing a wide range of deleterious effects (reviewed in Ref. 14). In exposed individuals, more than 80% of PCB metabolites in the lung are bound to CCSP (5, 15). In the kidney, CCSP mediates the uptake and accumulation of MeSO2-PCB in the proximal tubules by yet unknown receptor pathways (5). A central role of CCSP as a determinant of PCB bioaccumulation is further supported by the finding that no MeSO2-PCBs accumulate in the lungs or in the kidneys of CCSP-deficient mice (16).

As a further step in elucidating the molecular pathways regulating CCSP activity, we performed experiments to identify the cellular receptors that mediate the uptake of the protein into CCSP target tissues. We were able to uncover a two-receptor pathway responsible for the uptake of CCSP into the kidney and possibly other tissues. This pathway consists of cubilin, a peripheral membrane protein that binds CCSP, and an endocytic receptor megalin that associates with cubilin and mediates the endocytic uptake of cubilin·CCSP complexes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and General Methods-- Antibodies directed against rat CCSP (17), rabbit megalin (18), or rat cubilin (19) have been described before. Anti-beta 2-microglobulin IgG was purchased from DAKO (Hamburg, Germany). Mid-stream urine samples from patients with myeloma-associated Fanconi syndrome were generously provided by P. Aucouturier (Necker Hospital, Paris, France); urine samples from patients with Imerslund-Grasbeck syndrome were obtained from O. de Baulmy (Hopital Robert Debré, Paris, France). Experiments involving SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were performed according to standard procedures.

Protein Purification and Cell Uptake Studies-- Recombinant glutathione S-transferase (GST) or a fusion protein of glutathione S-transferase and rat receptor-associated protein (GST-RAP) were produced in DH5alpha bacteria as described before (20). Rat lavage CCSP and recombinant rat CCSP were purified as published previously (21, 22). The proteins were radiolabeled with 125I using the IODOGEN method (23). The cellular uptake and degradation of radiolabeled proteins were performed in Brown Norway rat yolk sac carcinoma cells cultured in standard medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum). Cellular degradation of 125I-ligands added to the culture medium was determined by standard protocols and expressed as nanograms of 125I-labeled trichloroacetic acid-soluble material released into the culture medium per mg of total cell protein (24).

Animal Studies-- For urine collection, megalin-deficient mice and control litter mates were placed in metabolic cages for 16 h and given 10% sucrose in drinking water. Urine samples (~5 ml/16 h) were collected on ice and were qualitatively indistinguishable from samples collected without sucrose load. Urine volume per h and creatinine levels were similar in megalin -/- and in control mice (~0.5 mmol creatinine/liter). For bronchio-alveolar lavage, mice were sacrificed by cervical dislocation, and the trachea was exposed and canulated. The diaphragm and parietal pleura were punctured to deflate the lungs. The lungs were slowly lavaged with 1 ml of sterile saline (0.15 M NaCl). The lavage was centrifuged at 10.000 × g for 10 min at 4 °C to remove alveolar macrophages and cellular debris. Samples were concentrated by centrifugation through a low binding cellulose membrane (Millipore) at 1.500 × g for 3 h at 4 °C. The concentration of the recovered proteins was determined, and samples were stored at -80 °C for further analysis.

BIAcore Analysis-- Binding of ligands to megalin or cubilin was quantified by BIAcore analysis (Biosensor, Uppsala, Sweden) as described (20). For generation of the receptor chips, a continuous flow of HBS buffer (10 mM HEPES, 3.4 mM EDTA, 150 mM NaCl, 0.005% surfactant P20, pH 7.4) passing over the sensor surface was maintained at 5 µl/min. The carboxylated dextran matrix of the sensor chip flow cell was activated by injection of 60 µl of a solution containing 0.2 M N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and 0.05 M N-hydroxysuccinimide in H2O. Then, 180 µl of 10 mM sodium acetate, pH 4.5, containing 10 µg/ml of purified rabbit megalin or rabbit cubilin was injected. The remaining binding sites were blocked by subsequent injection of 35 µl of 1 M ethanolamine, pH 8.5. The surface plasmon resonance signal from the immobilized receptors generated BIAcore response units equivalent to 30 fmol of megalin/mm2 and 40 fmol of cubilin/mm2. To test ligand binding, megalin or cubilin immobilized on the CM5 BIA sensor chip were incubated with the ligands in 10 mM HEPES, 150 mM NaCl, 1.5 mM CaCl2, 1 mM EGTA, pH 7.4, and the relative increase in response between megalin and control flow channels was determined. For determination of binding affinities, different concentrations of each ligand were subjected to BIAcore analysis on the cubilin sensor chip. The kinetic parameters were determined by using BIAevaluation 3.0 software.

Immunohistochemistry-- Wild type and megalin-deficient mice were perfusion-fixed through the heart with 4% paraformaldehyde in 0.1 M sodium cacodylated buffer. The collected tissues were trimed and immersion-fixed for 1 h in 1% paraformaldehyde, infiltrated with 2.3 M sucrose containing 2% paraformaldehyde for 30 min, and frozen in liquid nitrogen. Semithin 0.8-µm cryosections from the tissues were incubated with rabbit anti-rat CCSP antibody (1:1000 dilution) or rabbit anti-rat cubilin antibody (1:2000 dilution) for 1 h at room temperature, followed by either Alexa 546-conjugated anti-rabbit IgG (Molecular Probes, Leiden, The Netherlands) or peroxidase-conjugated anti-rabbit IgG (DAKO). For the latter incubations, peroxidase was visualized by diaminobenzidine, and the sections were counterstained with Meier's stain for 2 min. For electron microscopy, ultrathin 70- to 90-nm cryosections were incubated overnight at 4 °C with the anti-cubilin antibody (1:5000 dilution) followed by 10 nm of gold-conjugated anti-rabbit IgG (British BioCell International, Cardiff, United Kingdom).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study was conducted to identify the receptors responsible for cellular uptake and tissue accumulation of CCSP and CCSP·ligand complexes. We focused our attention on the proximal tubules of the kidney, because this tissue has been shown to clear CCSP and CCSP·PCB complexes from the glomerular filtrate (13, 16). Two efficient receptor pathways are known to operate in the epithelium of the renal proximal tubule (reviewed in Ref. 25). One is cubilin, a peripheral membrane protein involved in the clearance of filtered albumin (26). The other receptor is megalin, a multifunctional scavenging receptor of the low density lipoprotein receptor gene family. The latter receptor is responsible for retrieval of vitamin·carrier complexes from the glomerular filtrate. These complexes include 25-OH vitamin D3·vitamin D-binding protein and vitamin A·retinol-binding protein (20, 27).

To test whether cubilin or megalin may be responsible for tubular uptake of CCSP, we analyzed binding of CCSP to both receptors by BIAcore analysis. As seen in Fig. 1A, purified rat lavage CCSP bound to cubilin but not to megalin immobilized on the BIA sensor chips. The Kd of CCSP binding to cubilin was 56 nM (Fig. 1B). Similar results were obtained with recombinant rat CCSP (recCCSP); however, the affinity of binding was slightly lower (Kd of 400 nM, data not shown). We further investigated the requirements for CCSP binding to cubilin in more detail applying two inhibitors that interfere with ligand binding to this receptor. One inhibitor is EDTA, which chelates calcium ions required for cubilin/ligand interaction. As expected, binding of CCSP to cubilin was dependent on calcium and significantly reduced by the addition of EDTA to the assay medium (Fig. 2A). The second inhibitor tested was the receptor-associated protein (RAP), a cellular chaperone that antagonizes endocytic receptors including cubilin. Recombinant RAP can be applied exogenously to block binding of ligands to cubilin in vitro or in cells (28). Preincubation of cubilin with RAP impaired the ability of the receptor to interact with CCSP (Fig. 2B).


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Fig. 1.   BIAcore analysis of CCSP binding to cubilin and megalin. In panel A, cubilin and megalin immobilized on BIA sensor chips were incubated with purified rat lavage CCSP at a concentration of 1 µM. Binding of CCSP was observed to cubilin but not to megalin. In panel B, immobilized cubilin was incubated with the indicated concentrations of rat lavage CCSP. From the kinetic parameters, a Kd value of 56 nM for CCSP binding to cubilin was calculated (see "Experimental Procedures").


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Fig. 2.   Inhibition of CCSP binding to cubilin by EDTA and RAP. In panel A, binding of 0.5 µM recombinant rat CCSP to cubilin was tested in the absence (Cubilin) or in the presence of 20 mM EDTA (Cubilin + EDTA). In panel B, cubilin was pre-incubated with 10 µM RAP resulting in 400 response units. No further significant increase in response was achieved by subsequent addition of 0.5 µM recCCSP or 10 µM RAP, indicating inhibition of ligand binding by RAP. As a control, 0.5 µM recombinant CCSP were added to the receptor chip without prior addition of RAP (50 response units). The arrows denote the time points of addition of reagents to the BIA sensor chips.

So far, our studies had established specific and calcium-dependent binding of CCSP to cubilin, a peripheral membrane protein in the renal proximal tubule. Because tubular epithelial cells internalize CCSP in vivo (5), we investigated whether binding of CCSP to cubilin resulted in endocytic uptake of the protein. As a model cell line we used Brown Norway rat yolk sac carcinoma cells (BN16 cells), which express abundant amounts of cubilin and megalin (29). Thus, these cells represent the receptor profile of proximal tubular cells. When 125I-labeled recCCSP was added to BN16 cells, time-dependent uptake and lysosomal degradation of the protein were observed (Fig. 3). Uptake and degradation of CCSP were blocked by the receptor antagonist RAP and by chloroquine, an inhibitor of lysosomal degradation (Fig. 3). To confirm that the cellular catabolism of CCSP was dependent on cubilin activity, we applied anti-cubilin antibodies to block the receptor on BN16 cells. As controls, we used non-immune IgG or antiserum directed against megalin. As expected, addition of anti-cubilin IgG to the assay medium significantly inhibited CCSP degradation as compared with control IgG (Fig. 4, columns 1 and 2). Surprisingly, anti-megalin IgG also inhibited CCSP degradation to a similar extend (Fig. 4, column 3). Combined application of anti-cubilin and anti-megalin antibodies inhibited CCSP degradation even further (Fig. 4, column 5). The inhibition of CCSP degradation by anti-megalin antibodies was not because of a cross-reactivity of the antiserum with cubilin. As shown in Fig. 5, anti-megalin antiserum did not recognize this receptor in Western blot analysis of BN16 membrane preparations (Fig. 5).


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Fig. 3.   Degradation of recCCSP by BN16 cells. Replicate monolayers of BN16 cells received 0.5 ml of Dulbecco's modified Eagle's medium (without glutamine) containing 0.2% (w/v) bovine serum albumin and 230 ng/ml of 125I-recCCSP (specific activity 1754 cpm/ng). In addition, the medium contained either 100 µg/ml of GST-RAP (open circles), 50 µg/ml of GST (closed circles), or 200 µM chloroquine (open triangles). After incubation at 37 °C for the indicated periods of time, the amount of radiolabeled degradation products secreted into the medium was determined. Each value represents the mean of duplicate incubations.


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Fig. 4.   Inhibition of recCCSP degradation by anti-cubilin and anti-megalin IgG. Replicate monolayers of BN16 cells received 0.25 ml of Dulbecco's modified Eagle's medium (without glutamine) containing 0.2% (w/v) bovine serum albumin and 270-450 ng/ml of 125I-recCCSP (specific activity 1754 cpm/ng). In addition, the medium included 200 µg/ml of the indicated purified IgGs. After 2 h incubation at 37 °C, the amount of radiolabeled degradation products secreted into the medium was determined. Each value is the mean of four separate experiments (± S.E.). 100% values represent degradation rates in the absence of added immunoglobulins and range from 70-450 ng of CCSP/mg of cell protein.


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Fig. 5.   Western blot analysis of BN16 cell membranes. 10 (lane 1) or 1 µg (lane 2) of partially purified membrane proteins from BN16 cells were subjected to non-reducing 4-12% SDS-PAGE and transfer to nitrocellulose membranes. Replicate filters were incubated with rabbit anti-cubilin IgG (lane 1) or sheep anti-megalin IgG (lane 2). Bound IgGs were detected using chemiluminescence. The position of migration of marker proteins of 250 and 98 kDa in the gel is indicated. No cross-reactivity of the anti-megalin IgG with cubilin was observed (and vice versa).

Our studies had uncovered the finding that degradation of CCSP in BN16 cells is dependent on megalin, a receptor that does not bind CCSP directly. These results were intriguing as they supported a previous hypothesis about the mechanism of cubilin action. Cubilin is a protein without transmembrane or cytoplasmic domains that remains only loosely associated with the plasma membrane. Because the receptor is able to bind to megalin, it was proposed that cubilin may undergo endocytosis by association with megalin (30, 31). Thus, binding of inhibitory antibodies to megalin may affect ligand uptake via cubilin in BN16 cells. To confirm that such a dual receptor pathway is also operable in vivo, we analyzed the renal metabolism of CCSP in patients and in laboratory animals lacking cubilin or megalin. If our hypothesis was correct, kidneys with deficiency in either receptor should excrete CCSP. First, we tested urinary excretion of CCSP in patients with Imerslund-Grasbeck syndrome, an inheritable defect in the cubilin gene (32). As a control, we analyzed patients with renal Fanconi syndrome. These individuals suffer from a general tubular resorption defect and excrete numerous plasma proteins that are normally reabsorbed by the proximal tubules. As shown in Fig. 6, patients with Imerslund-Grasbeck syndrome excreted significant amounts of CCSP in their urine (lanes 2 and 4). Similar amounts of the protein were detected in urine of Fanconi patients (lane 5). No CCSP was observed in the urine of control subjects (lanes 1 and 3). The urinary loss of CCSP in patients with Imerslund-Grasbeck syndrome was not because of an unspecific tubular resorption defect, because beta 2-microglobulin, a marker of tubular dysfunction, was not excreted. This protein is taken up by other tubular receptor pathways and excreted in patients with Fanconi syndrome (Fig. 6). A total of four patients with Imerslund-Grasbeck syndrome, three patients with Fanconi syndrome, and six control subjects were analyzed. All samples gave the same results as the ones shown in Fig. 6. Next, we tested for excretion of CCSP in mice genetically deficient for megalin (20). Consistent with a role of this receptor in the renal uptake of CCSP, the animals secreted significant amounts of CCSP. The protein was not found in the urine of control animals (Fig. 7). A deficiency in the tubular uptake of CCSP in megalin knockout mice was further confirmed by immunohistochemical analysis of mouse kidney sections using anti-rat CCSP antiserum. In wild type mouse kidneys, abundant CCSP was detected in endosomal and lysosomal compartments of proximal tubular cells indicating uptake of the protein from the glomerular filtrate. No CCSP staining was observed in megalin-deficient kidneys (Fig. 8). To characterize the consequence of megalin deficiency on functional cubilin expression in more detail, we investigated the subcellular localization of this receptor in kidney sections. In wild type tissue, cubilin was present on the apical brush border surface of proximal tubular cells (Fig. 8). In particular, the protein was found in endosomes and recycling membrane vesicles, so-called dense apical tubules, which carry internalized receptors back to the cell surface. This pattern is consistent with a role for cubilin as recycling endocytic receptor (Fig. 9). In tubular cells lacking megalin, the total amount of cubilin found within the cells and on the brush border surface was dramatically reduced (Fig. 8). Small amounts of the protein found on the apical surface were present in membrane vesicles of significantly lesser density than dense apical tubules. These membrane vesicles most likely represented vesicles carrying newly synthesized cubilin from the trans-Golgi network to the cell surface. In these cells, cubilin was distinctly excluded from endosomes and dense apical tubules (Fig. 9).


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Fig. 6.   Urinary excretion of CCSP in patients with Imerslund-Grasbeck syndrome. Urine samples (5 µl) from two healthy control subjects (CTR; lanes 1 and 3), two patients with Imerslund-Grasbeck syndrome (IGS; lanes 2 and 4), and one patient with renal Fanconi syndrome (FS; lane 5) were subjected to 4-20% non-reducing SDS-PAGE and Western blot analysis using antisera directed against human CCSP or human beta 2-microglobulin (beta 2-M). Bound IgGs were detected using chemiluminescence.


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Fig. 7.   Urinary loss of CCSP in megalin-deficient mice. Urine samples (15 µl) from two wild type (+/+) and three megalin-deficient (-/-) mice were subjected to 4-20% non-reducing SDS-PAGE and Western blot analysis using anti-rat CCSP antiserum. Bound IgGs were detected using chemiluminescence. The position of migration of marker proteins in the gel is indicated.


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Fig. 8.   Immunohistochemical analysis of CCSP and cubilin in mouse kidneys. Detection of CCSP (upper panel) or cubilin (lower panel) in kidney sections of wild type (+/+) or megalin-deficient (-/-) mice is shown. The arrows in the upper panel indicate CCSP present in apical endosomes and lysosomes of wild type cells. Cubilin, ×1.400; +/+ CCSP, ×1.050; -/- CCSP, ×700).


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Fig. 9.   Electron microscopical analysis of cubilin expression in mouse kidneys. Electron micrographs of sections through proximal tubules of wild type (+/+; upper panel) or megalin-deficient kidneys (-/-; lower panel) are shown. Arrows in wild type tissue indicate dense apical tubules. Arrows in knockout tissue highlight membrane vesicles of lesser density, most likely transport vesicles originating from the trans-Golgi network. BB, brush border membrane; E, endosomes; ×70.000.

The studies described above have uncovered a dual receptor mechanism that mediates the cellular uptake of CCSP into the proximal tubules of the kidney. Next, we tested whether this receptor pathway may also be responsible for the catabolism of CCSP in the lung. Because no tissue was available from patients with Imerslund-Grasbeck syndrome, we focused our studies on the lungs of megalin-deficient mice. No obvious difference in the overall protein pattern (Fig. 10A) or the amount of CCSP (Fig. 10B) was detected in the bronchio-alveolar lavage of megalin -/- as compared with control mice. In addition, no significant difference in the interaction of total lavage proteins with megalin or cubilin was detected when samples from wild type or megalin-deficient mice were tested by BIAcore analysis (not shown). These results indicated that megalin-deficient lungs did not accumulate significant amounts of CCSP or other receptor ligands in the alveolar space. The findings, however, did not exclude the existence of alternative receptor pathways for catabolism of CCSP in the lung.


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Fig. 10.   Analysis of bronchio-alveolar lavage from wild type and megalin-deficient mice. A, bronchio-alveolar lavage fluid (5 µg of protein) from wild type (+/+; lanes 2, 3, 5, and 6) or megalin-deficient mice (-/-; lanes 1 and 4) were subjected to 4-15% reducing or non-reducing SDS-PAGE and staining with silver nitrate. The position of migration of marker proteins in the gel is indicated. B, duplicate samples as in panel A were characterized by Western blot analysis using anti-rat CCSP antiserum. No difference in the amount of CCSP was observed in wild type (lanes 2 and 3) as compared with megalin knockout samples (lane 1). The position of migration of marker proteins in the gel is indicated in kDa.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we have identified the receptors responsible for the uptake of CCSP into the proximal tubules of the kidney. The existence of such receptors has previously been postulated based on the uptake of CCSP and CCSP·PCB complexes into the kidney (5, 13, 16). This CCSP receptor pathway consists of the following two cellular proteins: cubilin, a peripheral membrane protein that binds CCSP, and megalin, an endocytic receptor required for endocytosis of cubilin·CCSP complexes.

Cubilin is a 460-kDa soluble protein. Structurally, it is characterized by the presence of 8 epidermal growth factor-type repeats and a cluster of 27 so-called CUB domains (30), a 110-amino acid motif first identified in components of the complement system (33). Cubilin was initially purified and cloned as the intrinsic factor receptor, a protein in the terminal ileum responsible for the uptake of intrinsic factor (IF)·vitamin B12 complexes (30, 34-36). Consistent with a crucial role of this protein in intestinal vitamin B12 absorption, patients with cubilin gene defects (Imerslund-Grasbeck syndrome) suffer from vitamin B12 deficiency and anemia (32). Apart from the terminal ileum, cubilin is also expressed in several other tissues, including the proximal tubules and the yolk sac (30, 31). This finding suggests that the protein may be involved in uptake of additional ligands in non-gastrointestinal cell types. In the proximal tubules, cubilin was recently shown to be responsible for clearance of filtered albumin (26). A role for the receptor in cellular uptake of high density lipoproteins was also demonstrated (37, 38). Given its function as an endocytic receptor, the structure of cubilin is unusual. The protein lacks obvious transmembrane or cytoplasmic domains and remains only loosely associated with the plasma membrane. Membrane attachment is mediated through an amphipathic helix located in the amino-terminal portion of the protein (28). Because cubilin is coexpressed with megalin and is able to bind to this receptor in vitro, a model was proposed whereby cubilin associates with megalin to recycle through the endocytic compartments (30). Several cubilin ligands have been suggested to utilize this coreceptor pathway, including the IF (30), albumin (26), and high density lipoproteins (31). However, no conclusive data have been described so far that such a coreceptor pathway is active in vivo. In the present study, we present novel experimental evidence that the cubilin/megalin coreceptor system exists in vivo and that it is responsible for the renal clearance of CCSP. Inhibition of cubilin in cultured cells (Fig. 4) or genetic inactivation in patients (Fig. 6) eliminates cellular uptake of CCSP. The same effect can be observed when megalin is inactivated (Fig. 4) or genetically deficient (Figs. 7 and 8).

The association of cell surface proteins with an endocytic receptor for recycling through the endocytic compartments has been described before. Most commonly, this phenomenon is observed for proteins linked to the plasma membrane via glycosyl phosphatidylinositol anchors. One example is the urokinase receptor, which associates with the low density lipoprotein receptor-related protein to achieve endocytic uptake of ligands (39). Both receptors associate with one another through their common ligand, the urokinase·plasminogen activator inhibitor-1 complex. The existence of endocytic coreceptors for the glycosyl phosphatidylinositol-anchored prion protein has also been postulated (40). The cubilin/megalin coreceptor system, however, is unique as it involves a peripheral membrane protein without glycosyl phosphatidylinositol anchor (cubilin) directly bound to an endocytic coreceptor (megalin). In the absence of megalin (as in megalin knockout kidneys), the total amount of cubilin expressed on the cell surface is reduced significantly (Figs. 8 and 9). The few receptor molecules on the apical membranes are unable to perform endocytosis of CCSP (Fig. 8) and seem to be excluded specifically from endosomes and recycling membrane vesicles (Fig. 9). These findings suggest the involvement of megalin in two distinct steps of cubilin transport and function. (i) Megalin is important but not essential for trafficking of cubilin through the secretory pathway to the plasma membrane. (ii) Megalin is absolutely required for internalization and endosomal targeting of cubilin. Taken together with data by Moestrup et al. (30) and Hammad et al. (31), a mechanism seems likely whereby cubilin remains associated with megalin throughout the endocytic pathway and on the cell surface. Ligands such as IF, albumin, or CCSP associate with this receptor complex by binding to sites on cubilin distinct from the megalin binding domain. Although the moderate affinity of cubilin for CCSP precluded the direct demonstration of a ternary complex between CCSP, cubilin, and megalin, the formation of such a complex with the IF has been reported before (30).

Expression of megalin and cubilin is not restricted to the proximal tubules in the kidney but seen in a number of embryonic and adult tissues. This raises the question whether the same receptor system may also be responsible for accumulation of CCSP in non-renal tissues. The unimpaired metabolism of CCSP in megalin-deficient lungs suggests that this receptor mechanism is not operable in the respiratory epithelium (Fig. 10). Besides the kidney, tissues with confirmed coexpression of megalin and cubilin are the yolk sac and the distal ileum (31). The cellular uptake of CCSP has been described in the renal proximal tubules and in the endometrium (13, 41, 42). It remains to be shown whether megalin and cubilin are coexpressed in the endometrium or whether CCSP is taken up in other tissues expressing both receptors.

The identification of a receptor pathway for clearance of CCSP from the glomerular filtrate raises some interesting hypothesis as to the physiological role of this protein in vivo. The main function of megalin in the proximal tubules is the retrieval of plasma carrier proteins that have been filtered through the glomerulus (reviewed in Ref. 43). These carrier proteins are the vitamin D-binding protein, the retinol-binding protein, transcobalamin, and transthyretin. The reabsorption of these carrier proteins is required to prevent the urinary loss of essential metabolites bound to the carriers including vitamins A, D3, and thyroxine (20, 27, 44). The clearance of CCSP by the same receptor pathway suggests that this protein may also act as a carrier for important metabolites that have to be scavenged from the renal filtrate. Consistent with this hypothesis, the CCSP homodimer has been shown to bind progesterone in a central pocket formed by the two protein subunits (45). Whether progesterone is a physiological ligand for CCSP in vivo remains unclear. Because the affinity for the steroid hormone greatly varies among CCSP species and is very low for human and monkey CCSP, the existence of yet unidentified CCSP ligands remains likely. Cubilin-deficient patients and megalin knockout mice should exhibit urinary loss of these metabolites. A function for CCSP in transport and cellular uptake of lipophilic substances is also supported by its role in bioaccumulation of MeSO2-PCB. Conceivably, these lipophilic xenobiotics displace the endogenous ligands transported by CCSP and thus are targeted to the same cell types that normally internalize CCSP·ligand complexes. Consistent with this hypothesis, mice deficient in megalin or patients defective for cubilin should still accumulate MeSO2-PCB in the lung but lack uptake into the kidney. At present, the poor viability of the megalin knockout mouse precludes the experimental proof of this hypothesis. However, the generation of a mouse model with kidney-specific megalin gene defects will enable us to test this concept.

In conclusion, our studies have uncovered the receptor pathway that is responsible for renal uptake of CCSP. These findings have elucidated an important step in the catabolism of the protein and will direct further research toward characterization of the metabolism and the functional significance of this carrier protein in vivo.

    ACKNOWLEDGEMENT

We are indebted to Hannelore Schulz, Charlotte Raeder, and Dana Bischof for expert technical assistance, to P. Aucouturier and O. de Baulmy for providing patient urine samples, and to C. Boensch for critical reading of the manuscript.

    FOOTNOTES

* The studies were funded by grants from the Deutsche Forschungsgemeinschaft, the Verbund Klinische Pharmakologie, Berlin-Brandenburg, the Norwegian Research Council, the Norwegian Cancer Society, and the Danish Medical Research Council. T. E. W. is a Heisenberg fellow of the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Contributed equally to this study.

¶¶ To whom correspondence should be addressed: Max-Delbrueck-Center, R.-Roessle-Strasse 10, D-13125 Berlin, Germany. Tel.: 49-30-9406-2569; Fax: 49-30-9406-2110; E-mail: willnow@mdc-berlin.de.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010679200

    ABBREVIATIONS

The abbreviations used are: CCSP, Clara cell secretory protein; PCB(s), polychlorinated biphenyl metabolite(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; RAP, receptor-associated protein; recCCSP, recombinant rat CCSP; IF, intrinsic factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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