Cubilin and megalin expression and their interaction in the
rat intestine: effect of thyroidectomy
Raghunatha R.
Yammani,
Shakuntla
Seetharam, and
Bellur
Seetharam
Division of Gastroenterology and Hepatology, Department of
Medicine, Medical College of Wisconsin and Zablocki Veterans
Administration Medical Center, Milwaukee, Wisconsin, 53226
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ABSTRACT |
Cubilin is a 460-kDa
multipurpose, multidomain receptor that contains an
NH2-terminal 110-residue segment followed by 8 epidermal growth factor (EGF)-like repeats and a contiguous stretch (representing nearly 88% of its mass) of 27 CUB (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domains. Cubilin binds to intrinsic factor (IF)-cobalamin (cbl, vitamin B12) complex and promotes the ileal transport of cbl. The
460-kDa form of cubilin is the predominant form present in the apical brush-border membranes of rat intestine, kidney, and yolk sac, but a
230-kDa form of cubilin is also noted in the intestinal membranes. In
thyroidectomized (TDX) rats, levels of intestinal brush-border
IF-[57Co]-labeled cbl binding, 460-kDa cubilin protein
levels and tissue (kidney) accumulation of cbl were reduced by ~70%.
Immunoblot analysis using cubilin antiserum of intestinal total
membranes from TDX rats revealed cubilin fragments with molecular
masses of 200 and 300 kDa. Both of these bands, along with the 230-kDa band detected in the total membranes of control rats and unlike the
460-kDa form, failed to react with antiserum to EGF. Mucosal membrane
cubilin associated with megalin was reduced from ~12% in control to
~4% in TDX rats, and this decreased association was not due to
altered megalin levels. Thyroxine treatment of TDX rats resulted in
reversal of all of these effects, including an increase to nearly 24%
of cubilin associated with megalin. In vitro, megalin binding to
cubilin occurred with the NH2-terminal region that
contained the EGF-like repeats and CUB domains 1 and 2 but not with a
downstream region that contained CUB domains 2-10. These studies
indicate that thyroxine deficiency in rats results in decreased uptake
and tissue accumulation of cbl caused mainly by destabilization and
deficit of cubilin in the intestinal brush border.
vitamin B12; transport
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INTRODUCTION |
GASTROINTESTINAL UPTAKE
AND TRANSPORT of dietary cobalamin (cbl, vitamin B12)
occur bound to gastric intrinsic factor (IF) by receptor-mediated
endocytosis via an apically expressed receptor for IF-cbl
(24). The receptor originally known as IF-cbl
receptor is now renamed cubilin due to the presence of 27 CUB
(initially found in complement components C1r/C1s, Uegf, and bone
morphogenic protein-1) domains that are contiguous and represent nearly
88% of its total mass. Upstream of the 27 CUB domains, cubilin
contains a 110-residue variable region, followed by eight EGF-like
repeats. This multidomain receptor (19) with a molecular
mass of 460 kDa binds to a variety of proteins including high-density
lipoprotein (HDL) (11), albumin (2), megalin,
and receptor-associated protein (19). Recently, it has
been demonstrated (10) that cubilin interaction with
megalin, a 600-kDa endocytic receptor, is essential for the endocytosis
of HDL in rat yolk sac carcinoma cells. Moreover, megalin expression in
these cells is suggested to be critical for cubilin expression at the
cell surface (10). Thus a close association of cubilin
with megalin in cultured cells may be important for both endocytosis
and their trafficking from the endoplasmic reticulum. Despite these
studies, it is not known whether a cubilin-megalin association exists
in the intact intestine, the site of dietary cbl absorption, and
whether such an association is important for cbl absorption and transport.
Previous studies (3) have shown that binding of IF-cbl to
the intestinal mucosa and cbl transport are decreased in TDX rats, and
a number of human studies (1, 7, 9) have demonstrated development of anemia in a majority of hypothyroid patients. Although the cause of anemia in these patients is not known, it is likely to be
due to the development of a cbl deficiency caused by malabsorption of
cbl. This hypothesis was tested in the present studies with the use of
thyroidectomized (TDX) rats as an experimental model. The results of
our study show that thyroxine deficiency in rats leads to impaired
uptake and transport of cbl due to poor expression of cubilin at the
cell surface. In addition, our study also demonstrates that, in TDX
rats, intestinal cubilin undergoes degradation, losing its
NH2-terminal region, including the EGF-like repeats, which results in its decreased ability to bind megalin.
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MATERIALS AND METHODS |
Materials.
The following were commercially purchased from the sources indicated:
[57Co]cbl (1.3 µCi/µg) and carrier-free
Na125I (ICN Radiochemicals, Irvine, CA). Affi-Gel 10, used
for coupling megalin, was purchased from Bio-Rad Laboratories
(Hercules, CA). The IF used in these studies was prepared from the rat
stomach, as described earlier (28). Megalin was purified
to homogeneity from rat kidney according to Kanalas and Makker
(13). Antiserum to purified megalin was raised in New
Zealand White rabbits. Rabbits were initially injected subcutaneously
at multiple sites with 50 µg of purified megalin mixed with complete
Freund's adjuvant, and after 2 wk, they were boosted with a total of
20 µg of megalin mixed with incomplete adjuvant. Antiserum to rat
renal cubilin was prepared as described earlier (29). A
polyclonal antiserum to human epidermal growth factor (EGF) raised in
rabbits was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
TDX rats, their respective sham-operated controls, and normal rats were
purchased from SASCO (Omaha, NE). Some TDX rats were treated with
intraperitoneal injections of thyroxine (1 µg/g body wt) daily for 6 days. Circulatory levels of total thyroxine in control, TDX, and
thyroxine-treated TDX rats were 4-5, 0.1-0.2, and 3.5-4
µg/100 ml, respectively. These data confirmed the thyroid status of
the different animal groups used in the study. A control group of rats
was treated with injections of 0.9% saline. Before the intestinal
tissue was harvested, rats in all three groups were first anesthetized
with phenobarbital (10 mg/kg body wt), their intestines were removed and chilled in ice-cold saline for 5 min, their luminal contents were
removed by washing with 5 ml of ice-cold saline, and mucosa was scraped
and homogenized in 10 mM Tris · HCl buffer.
cbl transport in vivo.
The in vivo intestinal uptake and kidney accumulation of
[57Co]cbl in control and TDX rats were carried out as
described earlier (23, 30). Briefly,
IF-[57Co]cbl (3.5 pmol) was directly instilled into the
stomachs of rats through a feeding tube. One hour after instillation of
the label, the animals were killed. The intestine was carefully removed from the pylorus to the ileocecal end and cut into segments. The segments were exposed to 5 mM KPO4 buffer, pH 5.0, containing 5 mM EDTA for 10 min to remove the surface-bound
radioactivity. The segments were then washed in phosphate-buffered
saline. The mucosa was scraped and counted to measure
[57Co]cbl uptake. Some animals were killed 6 h after
the instillation of ligand, and their kidneys were removed, rinsed with
ice-cold saline, blotted dry, weighed, cut into small pieces, and
counted for accumulated [57Co]cbl with the use of a
Beckman
-counter. The time frames of 1 h to study uptake of
IF-[57Co]cbl and 6 h to study renal accumulation of
[57Co]cbl are based on our earlier studies
(23). These earlier studies in rats have shown that, after
uptake of IF-cbl, cbl accumulates within the mucosa without cbl exiting
to the circulation, and at 6 h, mucosal cbl levels decline
significantly, and the cbl accumulated in the kidney is one-half of its
maximal level, which is reached between 12 and 24 h.
Membrane preparations.
Intestinal mucosa, kidney, or rat yolk sac (from 14-day-old pregnant
rats) was homogenized in a motor-driven Potter-Elvejhem homogenizer
using 10-15 strokes up and down in 10 mM Tris · HCl, pH
7.4, containing (in mM) 50 mannitol, 140 NaCl, 0.1 phenylmethlsulfonyl fluoride (PMSF), and 2 benzamidine (buffer A). The
homogenate was centrifuged at 100,000 g for 30 min, and the
pellet fractions were resuspended in the same buffer and used as total
membranes. Apical brush-border membranes from intestinal mucosa, yolk
sac, or kidney were prepared by the Ca2+ precipitation
method (14), as described earlier (29).
Immunoblotting.
Total and apical brush-border membranes (2-50 µg protein) were
subjected to nonreducing SDS-PAGE (4-7%). The separated proteins were then transferred overnight at 4°C onto Immobilion-P membrane by
use of a constant voltage of 30 V. The membranes were then probed with
diluted (1:5,000) antiserum to rat cubilin, megalin, or human EGF. The
immunoblots were quantified using the Ambis-radioimaging system, and
the intensity of the immunoreacting bands was translated into arbitrary
units. The linearity of the band intensity was confirmed with
immunoblots generated using pure rat renal cubilin (200-2,000 ng).
The immunoblots (Figs. 1, 2, and 5) are representative data from three
separate blotting experiments with membranes isolated from four to five
animals in each group.

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Fig. 1.
Isolated apical brush-border membranes from rat intestine
(25 µg protein), kidney, and yolk sac (2 µg protein) or intestinal
total mucosal membranes (10 µg protein) were subjected to nonreducing
SDS-PAGE (7%), transferred to Immobilion-P membrane, and probed with
diluted (1:5,000) polyclonal antiserum to rat cubilin and
125I-labeled protein A. The bands were visualized by
autoradiography.
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Fig. 2.
Apical brush-border membranes (A; 25 µg protein) and
total membranes (B; 50 µg protein) isolated from normal,
thyroidectomized (TDX), and thyroxine (T4) treated TDX rats
were subjected to nonreducing SDS-PAGE (7%) and transferred to
polyvinylidene difluoride membrane. Pure rat renal cubilin
(A; 500 ng protein) was also blotted as a reference protein.
The membranes were probed with diluted (1:5,000) antisera to rat
cubilin (A and B, lanes 1-3) or
to epidermal growth factor (EGF; B, lanes 4 and 5). The bands were visualized after treatment with
125I-labeled protein A by autoradiography.
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Megalin-cubilin interactions.
Isolated total membranes (100 µg protein) from control, TDX,
and thyroxine-treated TDX rats were incubated with
IF-[57Co]cbl (5 pmol) for 2 h at 22°C in 1 ml
containing 5 mM Tris · HCl buffer containing (in mM) 140 NaCl
0.1 PMSF, and 2 benzamidine. The membrane-bound radioactivity obtained
by centrifugation at 20,000 g was solubilized in 1 ml of
buffer A containing Triton X-100 (1%). The
Triton-solubilized fraction was incubated for 2 h at room
temperature with either preimmune serum or antiserum (5 µl) to rat
IF, cubilin, or megalin. The immune complex was precipitated with
protein A-Sepharose, and the [57Co]cbl bound to protein A
was measured using a Beckman
-counter.
Binding of NH2-terminal fragment of cubilin with
megalin.
The NH2-terminal fragment (which contains 110-amino acid
residue, NH2-terminal, eight EGF-like domains, and CUB
domains 1 and 2) and a downstream fragment containing CUB domains
2-10 were obtained by reverse-transcribed polymerase chain
reaction. The NH2-terminal fragment of cubilin was
amplified (2 kb) using the forward primer 5'-ATGTCCTCGCAGTTT-3',
corresponding to nucleotides 1-15, and the reverse primer
5'-AATGACTGCAGCAAAG-3', corresponding to nucleotides 1922-1936.
The CUB 2-10 region of the cubilin was amplified (2.6 kb) with
forward 5'-GTGGCGGCATCCTGA-3' and reverse 5'-ACTTTCAACTTCAAA-3'
primers, corresponding to nucleotides 1769-1783 and
4997-5011, respectively. Amplified fractions were purified by
means of a gel extraction kit (Millipore). The PCR products were
subcloned into the expression vector (pSec Tag B;
Invitrogen, Carlsbad, CA). The authenticity of the two cubilin
fragments amplified was confirmed by sequencing and by their ability to
bind the ligand IF-cbl and to react with polyclonal cubilin antiserum
(unpublished observations). The plasmid was translated in vitro
with the TNT quick-coupled transcription/translation system from
Promega (Madison, WI). The 35S-translated product was used
for binding to the affinity matrix containing bound rat megalin.
Other methods.
Total RNA from different groups was isolated according to Chomczynski
and Sacchi (6). Total RNA (25-50 µg) was used for blotting, and the blot was probed with a 32P-labeled,
2.1-kb cubilin fragment (NH2 terminus) generated by PCR.
Protein estimation in membrane samples was carried out according to
Bradford (4). Cubilin activity in the intestinal brush
borders was measured by its ability to bind the
IF-[57Co]cbl complex, as described earlier
(28). Briefly, rat IF-[57Co]cbl
(40-2,000 pg) was incubated with 25-50 µg of intestinal brush-border membrane protein in the presence of 10 mM
Tris · HCl buffer, pH 7.4, containing 5 mM of either
CaCl2 or Na2EDTA. The Ca2+ specific
binding of the ligand was calculated as before (26).
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RESULTS |
Cubilin protein expression and uptake of
IF-[57Co]cbl.
Immunoblot analysis of apical brush-border membrane isolated from
intestinal mucosa, kidney, and yolk sac revealed a predominant protein
band with a molecular mass of 460 kDa. However, in the intestine apical
membranes, another protein band, with a molecular mass of 230 kDa, was
identified; this band was very faint in the intestinal total membranes
but was absent in the apical membranes of kidney and yolk sac (Fig.
1). In addition to the 460-kDa band, a
very high molecular mass band that reacted with cubilin antiserum was
also detected in the kidney and yolk sac membrane, and the intensity of
this band was weak in the intestine. A faint band with a lower mass of
~200 kDa, which was not observed in the yolk sac membranes, could
also be seen in the intestinal and renal apical brush-border membranes.
When immunoblot analysis of the intestinal apical brush-border
membranes isolated from control and TDX rats was carried out (Fig.
2, top), the levels of the
460-kDa form of cubilin were drastically reduced, and in some rats it
was hardly identifiable. However, the intensity of the 460-kDa cubilin
band increased after treatment of TDX rats with thyroxine. To determine
whether the loss of cubilin expression in the apical brush border in
TDX animals was due to loss of total mucosal cubilin, immunoblotting
was carried out using higher amounts (50 µg) of total membrane
protein (Fig. 2, bottom). Both the 460- and the 230-kDa
forms of cubilin were identified in control rats (lane 1),
and in the total membranes of TDX animals, both of these protein bands
disappeared, and the molecular masses of immunocross-reactive bands
were 200 and 300 kDa (lane 2). Upon treatment with
thyroxine, the 460-kDa band reappeared and was the predominant band
(lane 3). These results suggest that, in thyroxine
deficiency, levels of the 460- and 230-kDa forms of cubilin are altered
due to their degradation. To confirm this and to examine the potential
degradation of cubilin from the NH2-terminal end, the total
membranes were subjected to immunoblot analysis with the use of EGF
antiserum (Fig. 2, bottom). Although the total membranes
from control rats reacted with EGF antiserum, demonstrating a single
band of 460 kDa (lane 4), this band was absent in membranes
obtained from TDX rats (lane 5).
When the immunoreactive cubilin bands observed in either intestinal
brush border or total membranes were quantified (Fig. 3), it was clear that TDX actually had no
significant effect on the total membrane cubilin protein levels (Fig.
3, bottom), but it decreased cubilin protein levels in the
apical brush border (Fig. 3, top). Furthermore, TDX had no
effect on either the IF-cbl binding activity of cubilin or cubilin
protein levels in the renal brush-border membranes of the rat (data not
shown). Taken together, these observations suggest that TDX affected
cubilin protein levels in the intestinal brush-border membrane and, as
such, had no effect on the total mucosal cubilin protein levels. This
observation is supported by dot blot hybridization, which showed that
altered thyroid status in rats had no significant effect on cubilin
mRNA levels (Fig. 4). Due to an extremely
low abundance of cubilin transcript in the intestine in general and in
rat intestine in particular, more sensitive quantitative Northern
blotting of cubilin mRNA levels was not possible.

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Fig. 3.
Immunoreactive bands shown in Fig. 2 were quantified by
the Ambis-radioimaging system. A: apical brush-border
membrane; B: total mucosal membranes. The data shown
represent means ± SD from 4 separate blots with the use of
different animals from each group.
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Fig. 4.
Total intestinal RNA (25 or 50 µg) from different group
of rats as indicated were blotted onto nitrocellulose membrane and
hybridized using a 32P-labeled amino-terminal cubilin cDNA
(2.1 kb) probe. The dots were visualized by autoradiography and
represent data obtained from dot blots by use of total RNA isolated
from 3 separate rats in each group.
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To further examine whether cubilin protein deficit in the intestinal
apical brush borders had indeed any effect on the
IF-[57Co]cbl binding activity, uptake, and plasma
transport of cbl, the ligand IF-[57Co]cbl was
administered orally, and the accumulation of [57Co]cbl in
the kidney was studied. The results (Table
1) show that, in vitro,
IF-[57Co]cbl binding activity in the brush border
decreased by ~70% from 66 to 20 fmol/mg protein. This decline in
IF-[57Co]cbl binding activity also resulted in a similar
decline in the in vivo uptake of IF-cbl from 60 to 18 fmol/mg protein
and of cbl accumulation in the kidney from 2.8 to 0.7 fmol/mg protein. These results clearly indicate that thyroxine deficiency results in
decreased intestinal mucosal uptake and tissue accumulation (kidney) of
orally administered cbl.
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Table 1.
Effect of thyroidectomy on intestinal brush border on
IF-[57Co]Cbl binding and in vivo mucosal uptake and
renal accumulation of [57Co]Cbl
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Megalin expression in the intestine and its interaction with
cubilin.
Earlier in vitro studies (19) had shown that purified
cubilin bound to megalin, and in cultured yolk sac cells, megalin association with cubilin was suggested to be important for the endocytosis of HDL and that synthesis of megalin was required for the
cell surface expression of cubilin (10). Thus we wanted to
examine first whether the cubilin-megalin association occurred in the
intact intestinal tissue and if so, whether this association was
affected in TDX animals. Our initial immunoblot studies using the
apical brush-border membrane (Fig.
5A) from the proximal and distal regions of the adult rat intestine revealed that megalin expression in rat intestine was limited to the distal regions. Furthermore, megalin levels in the distal intestinal apical
brush-border membranes (Fig. 5B) or in the total membranes
(Fig. 5C) did not change significantly in control
(lanes 1 and 4), TDX (lanes 2 and 5), or thyroxine-treated TDX rats (lanes 3 and 6). Similar to intestine, renal megalin levels also
did not change significantly in any of the three groups of rats (data
not shown).

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Fig. 5.
Megalin expression in rat intestine apical brush-border membranes
from normal rat proximal (P) and distal (D) intestine (50 µg protein;
A), distal apical brush-border membrane (25 µg protein;
B), or total distal intestinal membranes (10 µg protein;
C) from normal (lanes 1 and 4), TDX
(lanes 2 and 5), and thyroxine-treated
(lanes 3 and 6) TDX rats were subjected to
nonreducing SDS-PAGE (4%) and further processed for immunoblotting
using diluted (1:5,000) antiserum to rat megalin.
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Because cubilin association with megalin was suggested to be essential
for the endocytosis of ligands bound to cubilin, we initially examined
whether such an association exists in the intestinal mucosa membranes.
When the IF-[57Co]cbl radioactivity bound to total
membranes of the normal rat was extracted and treated with antiserum to
megalin, nearly 12% of the radioactivity was immunoprecipitated (Fig.
6, bar A). In TDX rats,
megalin-associated radioactivity declined to 4% (bar B), and upon thyroxine treatment of TDX rats, the total
membrane IF-[57Co]cbl radioactivity associated with
megalin rose to nearly 24% (bar C). These results clearly
indicate that the megalin-cubilin association exists in the native
intestinal membranes and that this association is affected in TDX rats.
When antiserum to IF or cubilin was used for immunoprecipitation
experiments, nearly 80-85% of the radioactivity extracted from
the apical brush-border membrane was precipitated.

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Fig. 6.
Total distal intestinal membranes (200 µg protein) from
control (A), TDX (B), and thyroxine-treated TDX
rats (C) were incubated with rat intrinsic factor
(IF)-[57Co]cobalamin (cbl) (5 pmol) for 2 h at
22°C. The membranes were then solubilized with TBS containing Triton
X-100 (1%). The solubilized extract containing 2-3 pmol of the
IF-[57Co]cbl was first treated with rat megalin antiserum
(10 µl), followed by treatment with a 1:1 suspension of protein
A-Sepharose (50 µl). After a 12-h incubation, the reaction mixture
was microfuged, the pellet was washed extensively with TBS, and the
radioactivity was measured. Values reported are means ± SD of 3 immunoprecipitation experiments by use of membranes from 4 animals in
each group.
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The observation that, in TDX rats, cubilin did not react with antiserum
to EGF (Fig. 2, bottom) and that its association with megalin was impaired suggested that the NH2-terminal region
of cubilin, including the EGF repeats and CUB domains 1 and 2, may be
involved in the interaction with megalin. To test this directly, cubilin cDNA fragments containing the 110-residue amino-terminal end
that also included the eight EGF-like repeats and CUB domains 1 and 2 and a downstream fragment containing CUB domains 2-10 were
translated in vitro. SDS-PAGE analysis of the labeled translated products (Fig. 7) revealed that, in both
cubilin fragments, the CUB 2-10 domain (lane 1) and the
translated NH2-terminal (lane 2) and the
molecular mass of the synthesized proteins, the 100- and 71-kDa values
were very close to the expected values of 110 and 74 kDa,
respectively. SDS-PAGE analysis (Fig. 7) of the radioactivity bound to megalin revealed that the amino-terminal fragment of cubilin
bound to megalin in the presence of Ca2+ (lane
3) but not in the presence of EDTA (lane 4). The CUB
2-10 fragment did not bind to megalin in the presence of either
Ca2+ (lane 5) or EDTA (lane 6).

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Fig. 7.
SDS-PAGE analysis of 35S-labeled proteins translated in
vitro using the reticulocyte lysate translation system and transcripts
from the CUB (initially found in complement components C1r/C1s, Uegf,
and bone morphogenic protein-1) 2-10 domains (lane 1)
and the NH2-terminal (lane 2) cubilin fragments.
The 35S-labeled NH2-terminal cubilin fragment
(lanes 3 and 4) and CUB 2-10 fragment
(lanes 5 and 6) recovered after binding to
megalin-affinity matrix in the presence of Tris · HCl buffer
containing either Ca2+ (lanes 3 and
5) or EDTA (lanes 4 and 6) were
subjected to SDS-PAGE (7%), and the protein bands were visualized
after fluorography. Data shown are representative of 4 different
translation and binding studies.
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DISCUSSION |
In this study, the presence of different molecular forms of
cubilin in the rat intestinal apical brush border and total membranes was first determined to assess the effect of TDX on their levels and
potential association with megalin. Earlier studies (reviewed in Ref.
25) had identified the molecular mass of intestinal cubilin in various species to be ~200 kDa. However, a recent study in
canine intestine (34) and the present studies in rat
intestine have identified the 460-kDa form of cubilin to be the
predominant form expressed in intestinal (Fig. 1) as well as in renal
and yolk sac brush-border membranes. In addition to the 460-kDa form, a
very high molecular mass band (Mr > 10
6) was also
noted in all of the rat tissue apical membranes. These observations are
in agreement with 1) the size of cubilin predicted on the
basis of its cDNA sequence (19) and 2) the
demonstration that, in vivo, cubilin is assembled as a noncovalent
trimer connected by an NH2-terminal coiled-coil helix
(16). Regarding the presence of a 230-kDa cubilin form in
the rat intestinal brush borders and its absence in kidney and yolk sac
membranes, several lines of evidence suggest that it is formed in the
intestinal brush border by the in situ action of extracellular
pancreatic proteases that are present in this tissue but are absent in
kidney and yolk sac. First, cubilin with a molecular mass of ~200 kDa
has also been observed in canine intestinal brush borders
(34). Second, in vitro studies have shown that trypsin
degrades bovine cubilin into several fragments (16).
Third, in metabolically labeled, polarized epithelial intestinal Caco-2
(22) and renal opossum kidney (21) cells that
lack proteases, a single cubilin band was noted in the apical
brush-border membrane. Fourth, cubilin purified from canine intestine
in the absence of proteolytic enzyme inhibitors generated several
functionally active fragments with a molecular mass <230 kDa
(27). Finally, extracellular proteolytic modification of
apical brush-border proteins is well known with other proteins
localized at this site (31). In addition to the 230- and
460-kDa forms of cubilin, a low-intensity band of ~200 kDa is also
observed in the apical bush border of intestine and kidney but not yolk
sac (Fig. 1), and a doublet band is seen around the 460-kDa band in the
intestinal apical brush border. The origin of these minor bands is not
known and could represent either a degraded product (200 kDa) or an
alternatively glycosylated form (<460 kDa), respectively. However,
because the doublet band was observed only in the intestinal but not in
the renal or yolk sac brush borders, it is likely that the doublet is
formed in the intestine apical brush border because of its initial
degradation soon after its delivery to this site. Additional studies
are required to establish the role of endogenous proteases in the
sequential degradation and turnover of cubilin.
Another interesting aspect of this study is the observation that the
levels of the 460-kDa form of cubilin declined (Fig. 2, top)
in the apical brush border of TDX rats but the total cubilin protein
levels (including all forms) present in the total membranes did not
(Fig. 3). This observation strongly suggests that the effects of TDX on
intestinal cubilin are posttranslational and is supported by the
demonstration of unaltered levels of intestinal cubilin mRNA (Fig. 4)
and renal brush-border protein levels (data not shown) in control and
TDX rats. In addition, cubilin fragments with molecular masses of 200 and 300 kDa were detected in the intestinal total membranes (Fig. 2,
bottom, lane 2) of TDX but not in control rats
(Fig. 2, bottom, lane 1). Moreover, EGF antiserum recognized only the 460- and not the 230-kDa form present in the control rats (Fig. 2, bottom, lane 4) or the 200- and 300-kDa forms present in the total intestinal membranes of the TDX
rats (Fig. 2, bottom, lane 5). Taken together,
these observations indicate that intestinal cubilin becomes unstable in
TDX rats and that its degradation to form the smaller-sized fragments
occurred from the NH2-terminal end that includes the
EGF-like repeats. Thus it is likely that the 230-kDa form of cubilin
may be a major intermediate during the degradative pathway of the
460-kDa form and that the degradative process may be initiated in the
brush borders in situ involving pancreatic proteases. Additional
studies will be required to determine the exact location of the site of
intestinal cubilin that generates the 230-kDa form in normal rat (Fig.
1) or its further degradation in thyroxine deficiency (Fig. 2,
bottom).
The effects of hypothyroidism on IF-[57Co]cbl binding to
brush border is specific to the intestinal tissue, as neither the
binding activity nor the cubilin protein levels were affected in the
rat kidney brush borders (data not shown). It is interesting to note that similar intestinal-specific changes have also been demonstrated during hypothyroidism for other proteins, such as carbonic anhydrase and Mg2+/HCO
ATPase (32).
In addition to intestinal-specific effects on some proteins, TDX also
causes other tissue-specific changes as noted with insulin-like growth factor receptor, whose activity in the anterior pituitary, but not in
brain, liver, and renal cortex (18), is affected. Thus tissue specificity of changes in specific protein levels or their membrane function in altered thyroid status may depend on whether the
effects are direct on the protein itself or whether they are indirect,
caused by TDX-induced changes in membrane fluidity. Thus the
possibility exists that the decreased plasma membrane transport of cbl
noted in this study could be due to TDX-induced changes in membrane
fluidity. This consideration is relevant, because it is well recognized
that altered thyroid status, particularly hypothyroidism in rats, is
known to increase the fluidity of both intestinal (5) and
renal (20) plasma membranes. However, this possibility is
highly unlikely for the following reasons. First, there is no evidence
that altered fluidity in the brush border increases the susceptibility
of cubilin to endogenous proteases. Second, on the basis of its
cDNA-predicted structure, cubilin has no transmembrane domain
(19); thus it is highly unlikely that lipid order changes
could affect its stability and activity. Third, the trans-cbl II
receptor, which facilitates the uptake of absorbed cbl from the
circulation bound to plasma trans-cbl II, is not altered in TDX rats
(3). Finally, TDX-induced posttranscriptional destabilization and modification are not unique to cubilin, as rat
liver glucocorticoid receptor has also been demonstrated
(15) to undergo similar changes in TDX rats.
The functional consequence of TDX due to cubilin deficit in the apical
brush border resulted in decreased mucosal uptake of the ligand
IF-[57Co]cbl and in subsequent accumulation of
[57Co]cbl in the kidney (Table 1). If decreased uptake of
IF-cbl and tissue (kidney) levels of cbl can be demonstrated with a
single oral dose of cbl, it is likely that, in the absence of thyroxine treatment, these animals may eventually develop cbl deficiency. This
suggestion is substantiated by a number of clinical studies that have
noted the development of anemia in adult (8), child, and
adolescent patients with hypothyroidism. Although these studies did not
address the cause of anemia in these patients, there is some evidence
from patients with autoimmune hypothyroidism (9) that
absorption of cbl, as determined by the Schilling test, was impaired.
It is likely that the development of cbl deficiency in these patients
is slow to develop and that the extent of the deficiency may depend on
the degree of hypothyroidism. Our studies (Fig. 2, top)
indicate that cubilin deficit or its recovery in the brush border upon
treatment with thyroxine may vary among rats.
Our immunoblot and immunoprecipitation studies show that, like cubilin
(30), megalin is also expressed in the distal intestine (Fig. 5A), a site of active endocytosis (33),
and that cubilin and megalin are associated in native tissue membranes.
Our data indicating an association of only 12% of cubilin with megalin in control rat intestinal mucosa may not be completely quantitative, because we were measuring only the immunoprecipitation of the ligand
IF-[57Co]cbl bound to the cubilin-megalin complex. It is
not known whether all of the antigenic sites of megalin were exposed
and thus were accessible for antibody recognition, particularly when it
exists as a complex with cubilin bound to the ligand. Despite this
uncertainty, when similar immunoprecipitation studies were carried out
with the total membranes from TDX rats and thyroxine-treated TDX rats (Fig. 6), the cubilin associated with megalin was 4 and 24%,
respectively. This clearly indicates that the modulation of cubilin
levels was responsible for the noted difference in the percentage of
its association with megalin, since megalin levels in either the
intestinal apical brush border or total membranes were not altered.
However, it is not clear at the present time why the percentage of
cubilin associated with megalin in thyroxine-treated rats rose to 24%, which is nearly twice the value of 12% noted in control animals. Because there was no significant effect of TDX or thyroxine treatment of TDX rats on megalin levels, it is likely that thyroxine treatment may affect steady-state cubilin levels by increasing its intracellular stability so that more cubilin would be bound to megalin.
Although our immunoprecipitation data clearly indicate the formation of
a megalin-cubilin-IF-cbl complex in the native apical intestinal
membranes, confirmation of these findings at the ultrastructural level
has been hampered due to technical difficulties. These include extremely low levels (fmol) of cubilin and IF bound to it
(12) and the difficulty of intrepretation of the data due
to the intense mucosal background staining obtained with the megalin
antibody (17). Despite this limitation, the association of
cubilin and megalin has been demonstrated by ultrastructural studies in
the apical invagination and microvilli of rat renal proximal tubule, yolk sac (19), and endocytic vesicles of yolk sac
endoderm-like cells (11), where the levels of these two
proteins are much higher relative to the intestinal tissue.
The observation that the NH2-terminal region, including the
eight EGF-like repeats and CUB domains 1 and 2, is involved in megalin
binding (Fig. 7) strongly suggests that disruption of the
cubilin-megalin interaction is due to loss of this region in intestinal
cubilin of TDX rats. To the best of our knowledge, this is the
first report that identifies the existence of a cubilin-megalin complex
in native intestinal membranes as well as the region of cubilin that
interacts with megalin. Additional studies are needed to further
dissect the role of individual regions of this fragment (i.e.,
NH2-terminal 110 residue, the eight EGF repeats, or CUB domains 1 and 2) that may be important in megalin binding. The loss of
this region due to destabilization of cubilin in TDX rats could result
in a loss of megalin binding and could occur both on the cell surface
after its delivery and within the cells. If it occurred
intracellularly, then failure to bind megalin might result in poor
trafficking of cubilin from the endoplasmic reticulum. Further studies
are needed to address these issues.
In conclusion, the results of our study show that, in TDX rats,
1) absorption and tissue accumulation of orally administered cbl is inhibited due to a deficit of cubilin in the apical brush borders; 2) cubilin is destabilized, causing its progressive
degradation; and 3) the cubilin-megalin association is
impaired due to loss of the NH2-terminal region (including
the EGF repeats), a region involved in megalin binding. Further studies
are required to address the site and cause of increased sensitivity of
intestinal cubilin in TDX rats.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the Department of Veterans
Affairs (7816-01P), awarded to B. Seetharam.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: B. Seetharam, MACC Fund Center, Rm. 6061, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail:
seethara{at}mcw.edu).
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.
Received 8 December 2000; accepted in final form 14 June 2001.
 |
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