A Two-receptor Pathway for Catabolism of Clara
Cell Secretory Protein in the Kidney*
Regina
Burmeister
§,
Inger-Margrethe
Bøe§¶,
Anders
Nykjaer
,
Christian
Jacobsen
,
Soeren K.
Moestrup
,
Pierre
Verroust**,
Erik I.
Christensen
,
Johan
Lund¶§§, and
Thomas E.
Willnow
¶¶
From the
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 
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 |
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 |
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 |
Materials and General Methods--
Antibodies directed against
rat CCSP (17), rabbit megalin (18), or rat cubilin (19) have been
described before. Anti-
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 DH5
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 |
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.
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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).
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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
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
2-microglobulin ( 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.
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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.
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 |
DISCUSSION |
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.
 |
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