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J. Biol. Chem., Vol. 276, Issue 48, 44777-44784, November 30, 2001
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From the Division of Gastroenterology and Hepatology, Departments
of Medicine and Biochemistry, Medical College of Wisconsin and the
Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee,
Wisconsin 53226
Received for publication, July 9, 2001, and in revised form, September 25, 2001
Using polymerase chain reaction-amplified
fragments of cubilin, an endocytic receptor of molecular mass 460 kDa,
we have identified two distinct ligand binding regions. Region 1 of
molecular mass 71 kDa, which included the 113-residue N terminus along
with the eight epidermal growth factor (EGF)-like repeats and CUB
domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of
CUB domains 6-8 bound both intrinsic factor-cobalamin (vitamin
B12; Cbl) (IF-Cbl) and albumin. Within these two
regions, the binding of both ligands was confined to a 110-115-residue
stretch that encompassed either the 113-residue N terminus or CUB
domain 7 and 8. Ca2+ dependence of ligand binding or
the ability of cubilin antiserum to inhibit ligand binding to the
113-residue N terminus was 60-65%. However, a combination of CUB
domains 7 and 8 or 6-8 was needed to demonstrate significant
Ca2+ dependence or inhibition of ligand binding by cubilin
antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to
the N-terminal cubilin fragment that included the eight EGF-like
repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both
regions of cubilin. Reductive alkylation of the 113-residue N terminus
or CUB 6-8, CUB 7, or CUB 8 domain resulted in the abolishment of
ligand binding. These results indicate that (a) cubilin
contains two distinct regions that bind both IF-Cbl and albumin and
that (b) binding of both IF-Cbl and albumin to each of
these regions can be distinguished and is regulated by the nonassisted
formation of local disulfide bonds.
The gastrointestinal uptake of cobalamin (vitamin B12;
Cbl)1 occurs bound to gastric
intrinsic factor (IF) by receptor-mediated endocytosis via a cell
surface receptor, intrinsic factor-cobalamin receptor (IFCR) (1). IFCR
isolated from canine ileal mucosa was estimated to have a molecular
mass around 220 kDa and bound IF-Cbl with high affinity (2), and
further proteolysis of this receptor revealed that IF-Cbl binding
occurred with a number of receptor fragments of molecular masses as low
as 80 kDa (3). In addition to the intestinal ileal mucosa, very high
levels of IFCR were also detected in mammalian kidney (4) and in rat yolk sac (5). In contrast to canine ileal mucosal IFCR, the purified
rat renal IFCR demonstrated a molecular mass of 457 kDa based on its
amino acid and amino sugar content. It bound 2 mol of IF-Cbl (4) and
like the yolk sac IFCR (5) was developmentally regulated (6). Although
the physiological significance for the high levels of IFCR expression
in the kidney was not known for a number of years, using proximal
tubular polarized epithelial opossum kidney cells it was demonstrated
that the apically expressed IFCR was able to internalize IF-Cbl and
mediate Cbl transport from the apical to basolateral medium (7). A
later study using a canine model with selective inherited intestinal
Cbl malabsorption syndrome (8) demonstrated that in these animals the
apical brush border membrane levels of IFCR in both ileal mucosa and kidney were depleted suggesting that the IFCR expressed in both tissues
was a product of the same gene.
Although it was thought for the last decade that the renal IFCR may
have some other function, its structure was only recently delineated
(9), and a number of subsequent studies have shown that the renal IFCR
is indeed a multifunctional receptor that is able to bind a variety of
protein ligands with differing affinities. These include IF-Cbl (1-2
nM) (4), albumin (0.63 µM) (10), high density
lipoprotein (0.1 and 56 nM) (11), and Based on the recent elucidation of its sequence, IFCR is now renamed
cubilin mainly because it consists of a contiguous stretch of 27 CUB
domains that represent nearly 88% of its total mass of 460 kDa and
because IF-Cbl binding has been shown to be localized to a region from
CUB domain 5 to CUB domain 8 (14). A CUB domain is a 110-115-amino
acid module present in developmentally regulated proteins that is known
to form a Despite these studies many aspects of the structure-function
relationship of cubilin are poorly understood, particularly its ability
to function as a multifunctional receptor, and the current studies were
directed in addressing some of these issues. The results of the current
studies show that cubilin contains two distinct ligand binding regions,
one the 113-residue N terminus and the other CUB domains 7 and 8 that
bind both IF-Cbl and albumin. Binding of both these ligands to each of
these regions can be distinguished and is regulated by the formation of
local disulfide bond(s) that form spontaneously in the absence of
molecular chaperones and exogenously added oxidized glutathione.
Materials--
Sepharose and rat serum albumin were purchased
from Sigma-Aldrich. pSec Tag B vector was from Invitrogen (Carlsbad,
CA), and canine pancreatic microsomes and the TNT quick coupled
transcription/translation system were from Promega (Madison, WI).
Fluoro-HanceTM used for autoradiography was obtained from
Research Products International Corp. (Mount Prospect, IL). Rat gastric
intrinsic factor used in the current studies was purified from the rat
stomach as described previously (15). Antiserum to purified rat renal cubilin raised in rabbits was prepared as described previously (4).
Polyclonal antiserum to human EGF raised in rabbits was purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Carrier-free
Na125I for iodination of rat serum albumin and
35S-Express protein labeling mix from PerkinElmer Life
Sciences. IODO-GEN was from Pierce.
Construction of Plasmids of Rat Cubilin Fragments--
Various
cubilin fragments were amplified by polymerase chain reaction (PCR).
The amplified products were subcloned into the expression vector pSec
Tag B. The following 13 constructs were subcloned and expressed:
N terminus (113 residues) (bp 3-14, bp 369-340), N-EGF (bp 3-14, bp
1404-1420), N-EGF + CUB 1-2 (bp 3-14, bp 2104-1221), CUB 1-4 (bp
4120-4136, bp 2777-2793), CUB 9-12 (bp 4171-4188, bp 5530-5556)
CUB 12-17 (bp 5212-5229, bp 7338-7353), CUB 18-27 (bp 7354-7369,
bp 10852-10869), CUB 5 (bp 2794-2808, bp 3124-3141), CUB 6 (bp
3142-3156, bp 3476-3492), CUB 7 (bp 3493-3507, bp 3816-3831), CUB 8 (bp 3832-3846, bp 4153-4170), CUB 7-8 (bp 3453-3507, bp
4153-4170), and CUB 6-8 (bp 3142-3156, bp 4153-4170). The sequence
of each PCR product obtained was confirmed to represent the correct
amplified sequence prior to its use. The plasmids were transcribed and
translated in vitro by TNT quick coupled transcription and
translation system from Promega. The 35S-translated
products were then used for further characterization.
Cell-free Translation and Processing of Cubilin
Fragments--
The constructs were transcribed and translated by the
TNT quick coupled system as described by the manufacturer. In some
experiments canine pancreatic microsomal membranes (1 µl) were added
to study the cotranslational processing and post-translational
modification of 35S-labeled rat cubilin fragments. To
confirm the ability of the added pancreatic microsomes to carry out
core N-glycosylation, mRNA encoding Preparation of Ligand Affinity Matrix and Ligand Affinity
Chromatography--
Rat serum albumin was coupled to CNBr-activated
Sepharose, and rat gastric IF was coupled to Cbl-Sepharose. One
milliliter of a 1:1 suspension in 10 mM Tris-HCl
buffer, pH 7.4, of the Sepharose-linked ligands (albumin or IF-Cbl) was
capable of binding to at least 500-700 ng of purified renal cubilin.
The 35S-translated cubilin fragments (50,000-75,000 dpm)
were incubated with 500 µl of a 1:1 suspension of Sepharose beads in
10 mM Tris-HCl, pH 7.4, containing 10 mM
CaCl2 or 10 mM EDTA. In some experiments, the
labeled cubilin fragments were preincubated with 2-5 µl of polyclonal antiserum to either cubilin or EGF. For experiments shown in
Figs. 7 and 8 the addition of 5 µl of the polyclonal antiserum to
cubilin and EGF resulted in maximum inhibition of ligand binding, and
the addition of a higher amount (10-25 µl) of the antisera did not
inhibit ligand binding further. Using similar amounts (50,000 dpm) of
protein synthesized from constructs CUB 7, CUB 8, or CUB 6-8, 5 µl
of antiserum was sufficient to precipitate >90% of all three labeled
proteins. Competition of ligand binding was carried out similarly
except that the labeled cubilin fragments were preincubated for 60 min
at 22 °C with either rat IF-Cbl (50 ng) or rat serum albumin (100 ng) prior to their binding to Sepharose linked to albumin or rat
IF-Cbl, respectively. Following binding for 60 min, the beads were
exhaustively washed with 10 mM Tris-HCl, pH 7.4, containing
140 mM NaCl (15-20 ml), and radioactivity bound was then
released by boiling the beads with SDS sample buffer and subjected to
nonreducing SDS-PAGE.
SDS-PAGE and Fluorography--
35S-Labeled
translation products or the ligand affinity-purified cubilin fragments
were subjected to nonreducing SDS-PAGE (12%). Gels were fixed and then
treated with Fluoro-HanceTM for about 30 min (as described
by the manufacturer), and the bands were visualized by fluorography. In
some experiments, the labeled cubilin fragments were first reduced with
2-mercaptoethanol (2%) and then alkylated with
N-ethylmaleimide (1 mM) prior to SDS-PAGE. The
bands were quantified by the AMBIS radioimaging system. SDS-PAGE data
shown in Figs. 2-4 and Figs. 11 and 12 are typical representations of
at least three separate translation and ligand binding experiments and
reductive alkylation experiments, respectively.
Other Methods--
Rat renal apical membranes were isolated by
the Ca2+ aggregation method as described previously (4).
The Ca2+-dependent binding of ligand,
IF-[57Co]Cbl (100-2500 pg), or 125I-rat
serum albumin (10-200 ng) (specific activity, 200,000 dpm/µg of
albumin) was determined using the rat renal membranes (25-50 µg of
protein). The total ligand bound in the presence of 10 mM Tris-HCl buffer, pH 7.4, containing 10 mM CaCl2
was subtracted from that bound in the presence of the same buffer but
containing 10 mM EDTA to obtain Ca2+-specific
ligand binding. Sequence alignment of the N-terminal region and CUB
domains 7 and 8 were performed by Jellyfish version 1.4 using
matrix-Gonnet.
In Vitro Transcription/Translation and Ligand Binding of Reverse
Transcriptase PCR-generated Cubilin Fragments--
The various
regions of cubilin that were amplified by reverse transcriptase PCR are
shown in Fig. 1. Initially all cubilin fragments were in vitro transcribed and translated to test
for their ability to synthesize a functional protein. Earlier studies (14) had revealed that when the media collected from stably transfected cells were tested for IF-Cbl binding, only the media from
cells transfected with CUB domains 5-8 bound IF-Cbl, and none of the
other cubilin fragments demonstrated IF-Cbl binding activity. In our
studies (Fig. 2), four cubilin fragments
encompassing CUB domains 1-4, 9-12, 12-17, and 18-27 were
efficiently translated in vitro (lanes 1) and
produced major proteins of molecular masses 45, 47, 70, and 100 kDa,
respectively, consistent with the number of CUB domains present in each
fragment. Each CUB domain consists of 110-115 residues with an average
polypeptide mass of around 110 kDa. The
[35S]methionine-labeled proteins synthesized by these
cubilin fragments failed to bind either IF-Cbl (lanes 2) or
albumin (lanes 3). In contrast, the ~71-kDa cubilin
fragment synthesized (Fig. 3A,
lane 1) from the N-terminal fraction, which contained the
113-residue N-terminal region along with the eight EGF-like repeats and
CUB 1-2 demonstrated Ca2+-dependent binding of
both IF-Cbl (Fig. 3B, lane 1) and albumin (Fig.
3C, lane 1). Within this region, the 113-residue
N-terminal region, which synthesized a protein of ~18 kDa
(Fig. 3A, lane 2) was by itself enough and
sufficient to bind both IF-Cbl and albumin (Fig. 3, B and
C, lanes 2). The cell-free translation of the
113-residue N terminus resulted in the synthesis of a predominant 18-kDa band, but there were three other bands of higher molecular mass
(36, 39, and 44 kDa). These higher molecular mass forms may represent
the aggregated forms of the 18-kDa form, and the size difference
between them could be due to different amounts of Triton X-100 bound to
them. This conclusion is based on the observation that all three forms
bound both ligands. Prior incubation of the translated product with
Sepharose alone did not eliminate these bands from the fraction that
was eluted from ligand affinity matrix (data not shown). The DNA
fragment encoding the eight EGF-like repeat region synthesized a
protein of 45 kDa but did not bind either of the two ligands (data not
shown). Since our earlier data (Fig. 2) had demonstrated that no ligand
binding activity occurred with cubilin fragments synthesized downstream
of CUB domain 9 and CUB domains 1-4, attention was focused to test
ligand binding with cubilin fragments synthesized by CUB domains 5-8. While all of the individual CUB domains between 5 and 8 were able to
synthesize protein products of molecular masses between 17 and 20 kDa
(Fig. 4A), only the proteins
synthesized with CUB 7 and CUB 8 were able to bind both IF-Cbl (Fig.
4B) and albumin (Fig. 4C). In addition, protein
products of molecular mass 28 or 37 kDa synthesized from CUB 7-8 or
6-8, respectively, also bound both the ligands (Fig. 4, B
and C). Lack of ligand binding (Fig. 4, B and
C) observed by protein synthesized by CUB 5 or CUB 6 was not
due to low levels of protein expressed by these constructs since the
amount of 35S radioactivity used for affinity ligand
binding was 3-4 times higher than that used for SDS-PAGE (Fig.
4A) analysis of the translated products.
The two ligand binding regions of cubilin, the 113-residue N terminus,
which included the eight EGF-like repeats along with CUB domains 1 and
2, and CUB domains 6-8 region contained three and five potential
N-glycosylation sites, respectively. Thus, we wanted to test
whether these sites are utilized for N-glycosylation and if
so, whether glycosylation at these site(s) affects ligand binding.
Thus, these constructs were translated in vitro in the presence and absence of canine pancreatic microsomes. SDS-PAGE analysis
(Fig. 5) failed to detect any shift in
the electrophoretic mobility (Fig. 5A) with either region 1 representing the N terminus + eight EGF-like repeats + CUB domains 1 and 2 (Fig. 5A, compare mobility in lanes 3 and
4) or region 2 containing CUB domains 6-8 (Fig.
5A, compare mobility in lanes 1 and
2). Moreover, both regions of cubilin bound the ligands
whether they were synthesized in the absence (Fig. 5, B and
C, lanes 1) or the presence (Fig. 5, B
and C, lanes 2) of pancreatic microsomes. Taken
together, these observations suggested that under the experimental
conditions used in the present studies these sites are not
N-glycosylated and that the ligand binding is not affected
when cell-free translation was carried out with the cotranslational
addition of canine pancreatic microsomes. However, a positive control
incubation to test the activity of the pancreatic microsomes indicated
that under similar experimental conditions, Saccharomyces
cerevisiae Ca2+ Dependence of Ligand Binding--
The binding of
IF-Cbl to its receptor is Ca2+-dependent, but
it is not known whether the dependence on Ca2+ for ligand
binding is a property of the full-length receptor or can be
demonstrated with functionally active regions of cubilin. To address
this issue, the binding of 35S-labeled cubilin fragments
were tested for Ca2+ dependence of binding to both IF-Cbl
and albumin (Fig. 6). The binding of
IF-Cbl and albumin to native renal brush border membrane was inhibited
by EDTA by 97 and 50%, respectively. EDTA-inhibitable binding of
IF-Cbl and albumin to region 1 encompassing only the 113-residue
N-terminal region of cubilin was 64 and 70%, respectively. Interestingly, the inclusion of eight EGF-like repeats with the 113-residue N terminus resulted in the abolishment of Ca2+
dependence for albumin binding, but the binding of IF-Cbl was 50%
Ca2+-dependent. In contrast, both CUB domains 7 and 8 bound IF-Cbl and albumin but demonstrated only 5-8%
Ca2+ dependence for the binding of both ligands. Although
the total ligand bound (Fig. 6, dotted line) did not
significantly change, CUB 7-8 or CUB 6-8 demonstrated an increased
Ca2+ dependence for both ligands.
Ca2+-dependent binding of both IF-Cbl and
albumin to CUB 7-8 was about 50%. While inclusion of CUB 6 further
increased the Ca2+ dependence of IF-Cbl binding by an
additional 40-45% to almost 95%, it had no additional effect on
Ca2+ dependence of albumin binding by CUB 7-8.
Effect of Cubilin and EGF Antisera on Ligand
Binding--
Polyclonal antiserum to rat renal cubilin was able to
inhibit by 58-64% (Fig. 7) IF-Cbl and
albumin binding to the 113-residue N terminus. However, it inhibited by
10, 25, and 60% the binding of IF-Cbl to CUB 7, CUB 8, and CUB 6-8,
respectively. On the other hand, the binding of albumin to CUB 7 and
CUB 8 was inhibited between 55 and 60% and reached 95% for CUB 6-8
(Fig. 7). Studies with antiserum to EGF (Fig.
8), which is relevant to only region 1, revealed inhibition of albumin binding by nearly 90%, while it had
very little or no effect on IF-Cbl binding to this region.
IF-Cbl Inhibits Albumin Binding at Both Regions of Cubilin--
To
examine whether the two protein ligands compete for binding, binding of
one ligand was carried out after a preincubation of the
35S-labeled cubilin fragments with the other ligand.
Preincubation with excess albumin did not inhibit the binding of IF-Cbl
by either the 113-residue or the CUB 6-8 fragment (Fig.
9). On the other hand, when similar
experiments were carried out with preincubation in the presence of
IF-Cbl, the binding of albumin by both regions of cubilin was inhibited
by >90-95%.
Local Disulfide Bond Formations in the Functional
Regions of Cubilin Are Important for Ligand Binding--
Sequence
analysis of the 113-residue N-terminal ligand binding region of cubilin
revealed two cysteine residues (Fig.
10) suggesting the potential formation
of only one disulfide bond in this region. On the other hand, sequence
alignment of the individual CUB domains 7 and 8 revealed (Fig. 10) the
presence of four cysteine residues in each one of these CUB domains
present at identical positions.
To test whether local disulfide bonds were formed following translation
of the two functional regions of cubilin and to further examine whether
the disulfide bonding is required for ligand binding, the labeled
translated fragments were subjected to reductive alkylation and ligand
binding. SDS-PAGE analysis (Fig. 11)
revealed that following reductive alkylation, the electrophoretic
mobility of the translated 113-residue N-terminal region decreased
(lane 2) relative to the unreduced sample (lane
1). In addition, while the nonreduced N-terminal protein bound
both IF-Cbl (lane 3) and albumin (lane 5),
reduction of the only disulfide bond formed between Cys-63 and Cys-117
destroyed binding of both ligands (lanes 4 and
6). Formation of a disulfide bond(s) as revealed by
decreased electrophoretic mobility was also evident with the cubilin
fragment encoded by CUB 6-8 (lanes 1 and 2).
Reductive alkylation inactivated binding of both IF-Cbl (lane
4) and albumin (lane 6) relative to the binding
obtained with the nonreduced forms (lanes 3 and
5). Reductive alkylation of these two cubilin regions
synthesized in the presence or absence of canine pancreatic microsomes
also revealed an identical shift in the mobility of the reduced forms
of cubilin fragments (data not shown).
To further confirm whether the loss of ligand binding due to disruption
of disulfide bonding in region 2 (CUB 6-8) is due to loss of intra- or
inter-CUB domain disulfide bonds, reductive alkylation of individual
CUB domains was carried out. SDS-PAGE analysis of the labeled proteins
revealed (Fig. 12) that CUB domains 7 and 8, which bind the ligand, demonstrated disulfide bonding, and upon
reductive alkylation, ligand binding to both these CUB domains was
completely inhibited (data not shown). However, CUB domains 5 and 6, which do not bind the ligand (Fig. 4), revealed the presence of
disulfide bonding in CUB 5 but not CUB 6. These observations indicated
that intramolecular disulfide bonds within CUB 7 or CUB 8 are important
for ligand binding.
Cubilin is a 460-kDa multidomain, multifunctional endocytic
receptor expressed in the apical membranes of tissue epithelial cells
and functions synergistically (16) with megalin, another endocytic
receptor of molecular mass 660 kDa. For an endocytic receptor, cubilin
is unique in that it has no discernable transmembrane domain (9), and
it is not fully understood how cubilin interacts with the apical lipid
bilayer membrane to expose its ligand binding sites to allow binding of
water-soluble ligands such as IF-Cbl and albumin.
The identification of two ligand binding regions localized to the first
one-third of the cubilin molecule (Fig. 2-4) suggested strongly that
in the context of full-length cubilin both these regions must be
outside the lipid bilayer so that they are able to bind the
water-soluble ligands. One of the regions of cubilin identified in this
study, the 113-residue N terminus, binds both IF-Cbl and albumin (Fig.
3). The N terminus of cubilin has also been demonstrated to form
coiled-coil In addition to the N terminus, there is evidence that the entire
cubilin molecule is peripherally localized to the apical lipid bilayer
at multiple sites with a variety of membrane interactions that could
include protein-lipid, protein-protein, and divalent cation-dependent. With this type of topography, it is very
likely that both ligand binding regions are exposed for ligand binding. These conclusions are based on a number of observations obtained from
different studies. These observations include (a)
partial solubilization of canine intestinal cubilin bound to either
native brush border or synthetic lipid vesicles by phospholipases,
detergents, and proteases (19), (b) partitioning of
intestinal cubilin in the detergent-poor aqueous phase following
treatment of apical brush border membrane with Triton X-114 (20),
(c) partial solubilization of native renal membrane-bound
cubilin with a number of reagents that included EDTA, heparin, and
phosphatidylethanolamine (9, 17), (d) full-length purified
renal cubilin of molecular mass 467 kDa bound to Triton X-100 micelles
bound 2 mol of IF-Cbl/mol (4) in a
Ca2+-dependent manner, and (e) the
functional topography of either native brush border- or lipid
vesicle-bound cubilin is the same in that the ligand binding and
antigenic sites of cubilin are exposed outside the lipid bilayer
(19).
Another important consideration of these studies is the observation
that the two functional regions of cubilin identified in this study are
able to bind the ligand when translated in vitro in the
presence or absence of pancreatic microsomes (Fig. 5, B and
C). Thus, it is unlikely that the potential
N-glycosylation sites present in these regions are utilized
for N-glycosylation, and the lack of core
N-glycosylation may not influence localized folding of the
functional regions of cubilin to affect ligand binding. Although
estimating the number of N-linked sugars based on
electrophoretic mobility shift in proteins with larger molecular mass
is not accurate, earlier studies of rat (6) and opossum (21) renal
cubilin have shown that cubilin is indeed N-glycosylated, and a more recent estimate of the number of N-linked sugars
suggests that rat renal cubilin may be N-glycosylated at
28-30 of 42 potential sites (9). Thus, it is possible that the 12-14
potential N-glycosylation sites that are not utilized may
also include the potential sites present in the two ligand binding
regions of cubilin. Alternatively, in these shorter cubilin fragments,
unlike in the full-length cubilin, the potential
N-glycosylation sites may not be accessible for
N-glycosylation, particularly considering that the cubilin constructs used did not contain the N-terminal signal sequence that is
essential for the recognition of the protein synthesized by the
endoplasmic reticulum membranes.
Although the two distinct ligand binding regions of cubilin are
confined to 110-113 residue units, their Ca2+ dependence
for ligand binding appear to be influenced by the flanking regions.
This is particularly so in region 2 (Fig. 6) where the presence of CUB
domain 6 increased Ca2+ dependence but not the total IF-Cbl
binding of the CUB 7-8 fragment. However, the inclusion of CUB domain 6 did not further enhance the Ca2+-dependent
binding of albumin by this cubilin fragment. In addition, the
Ca2+-dependent binding of both IF-Cbl and
albumin rose dramatically when both CUB domains 7 and 8 were present
together rather than being present as individual domains. These
observations suggested that there may be multiple Ca2+
binding sites localized both near and away from the ligand binding sites in CUB domains 7 and 8. The location, number, and nature of the
Ca2+ interaction may influence whether the Ca2+
requirement is absolute or obligatory, and further studies are needed
to identify the Ca2+ binding sites of the cubilin
fragments. One region of cubilin that may contain potential
Ca2+ binding sites are the EGF-like repeats as these
structures, present in many proteins (22), have been implicated in
Ca2+ binding. While both IF-Cbl and albumin binding to this
region demonstrated 60-70% Ca2+ dependence, inclusion of
all the EGF-like repeats with the 113-residue N terminus actually
eliminated Ca2+ dependence of albumin but not IF-Cbl
binding (data not shown). Thus, Ca2+ dependence of ligand
binding to cubilin could be regulated by the location of the
Ca2+ binding loop structure that is formed, and it is
likely that the position of these loops is different depending on the
ligand of choice.
In many ways the two functional regions of cubilin identified in this
study share many of the same properties as the full-length protein.
These include Ca2+-dependent ligand binding
(Fig. 6), the ability of cubilin antiserum to inhibit the binding of
both ligands (Fig. 7), and the ability of IF-Cbl to inhibit albumin
binding but not the ability of albumin to inhibit IF-Cbl binding (Fig.
9). It is interesting to note that there is a ~500-750-fold
higher affinity for IF-Cbl binding relative to albumin binding by the
purified full-length cubilin, and this appears to be true at both
functional regions of cubilin as well (Fig. 9). Ligand binding to the
N-terminal region that also included the EGF repeats could be
distinguished as the EGF antiserum was able to inhibit binding of
albumin by >90%, while it had no effect on IF-Cbl binding (Fig. 8).
Thus, it is likely that within each ligand binding region of cubilin,
the albumin and IF-Cbl binding sites are spatially related, yet they
can be distinguished. However, additional studies are needed to fully understand the spatial relationship of multiple ligands binding to
these functional units of cubilin and also how its nonfunctional units
influence the Ca2+ dependence of ligand binding.
Sequence comparison (Fig. 10) of the ligand binding regions of cubilin
revealed only 7% identity between the N-terminal region and CUB
domains 7 and 8. While the N terminus had between 12-14% identity
with either CUB domain 7 or 8, the two CUB domains by themselves had
close to 30% identity. CUB domain 8 contained a proline residue at
the19th position (corresponding to codon 1297 in the full-length
cubilin) that has been implicated in the high affinity binding of
IF-Cbl (23). Mutation of this proline to leucine resulted in a 5-fold
decreased affinity for IF-Cbl. Moreover, the mutation, P1297L appears
to be specific and is present in some (Finnish) but not other
(Norwegian and Saudi Arabian) patients with hereditary megaloblastic
anemia (24). It is interesting to note that a proline residue is also
present at a similar position in CUB domain 7 and in N terminus. In
addition, a proline residue is also conserved at the 85th position
(Fig. 10) in all three ligand binding regions. It is not known whether
the proline residue at the 19th position in the N terminus or CUB
domain 7 or the proline conserved in all three regions at position 85 has any role in ligand binding. In a canine model of inherited Cbl
malabsorption syndrome (8), the cubilin defect was due to its
misfolding that resulted in the failure of cubilin delivery from the
endoplasmic reticulum to the apical membranes. It is obvious that much
needs to be learned about the molecular defects of cubilin that cause defective uptake of IF-Cbl in the intestine or of albumin in the proximal tubular cells that result in the development of Cbl deficiency and proteinuria, respectively. Although proteinuria is often a common
finding in patients with Cbl malabsorption syndrome (25), some patients
do not develop proteinuria (26, 27). These studies indicate that
different mutations of the cubilin molecule may exist that affect
uptake of IF-Cbl, albumin, or both.
The formation of a disulfide bond and its importance in ligand binding
is evident in both ligand binding regions of cubilin. In the
113-residue N terminus, reductive alkylation resulted in the formation
of an extended form with lower electrophoretic mobility on SDS-PAGE
(Fig. 11). This bond is formed between Cys-63 and Cys-117 as these are
the only two cysteine residues present in this ligand binding region
(Fig. 10). Alternatively, in the second ligand binding region there is
also evidence of disulfide binding within CUB domains 6-8, formation
of which is essential for ligand binding (Fig. 11B). Within
this region, intra-CUB domain disulfide bonding appears to be important
as both CUB 7 and CUB 8 appear to form disulfide bonds (Fig. 12) that
are important for ligand binding (data not shown). It is interesting to
note that the functional cubilin fragments synthesized are able to form
disulfide bonds in the absence of cotranslationally added pancreatic
microsomes or oxidized glutathione. Addition of both these components
cotranslationally has been implicated in the formation of disulfide
bonds and cotranslational translocation of newly synthesized proteins
(28, 29) such as the 46-kDa mannose 6-phosphate receptor (30). The
spontaneous formation of functionally correct disulfide bonds in the
absence of added pancreatic microsomes suggested that the folding of
the mature cubilin may be hierarchal proceeding from secondary
structure via subdomains and domains toward the complete tertiary
structure. Local folding required for the acquisition of ligand binding
of cubilin fragments may be rapid, not needing the assistance of molecular chaperones such as protein disulfide isomerase, which is
known to assist in protein folding and disulfide bond formation (31).
In addition, the spontaneous formation of correct disulfide bonds in
cubilin may aid in the early acquisition of ligand binding as well as
its rapid vectorial delivery to the plasma membrane. Previously studies
(32) have shown that cubilin expressed in opossum kidney cells is very
rapidly transported to the plasma membrane with a
t1/2 of 30 min.
In summary, we have demonstrated that cubilin, a multifunctional
receptor, is able to bind both albumin and IF-Cbl at two distinct
regions. Within each region the binding of these two ligands occurs at
spatially related sites and is dependent on disulfide bonds formed
within each of these regions.
*
This work was supported by Department of Veterans Affairs
Grant 7816-01P (to B. S.).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.
§
To whom correspondence should be addressed: MACC Fund Center, Rm.
6061, Medical College of Wisconsin, 8701 Watertown Plank Rd.,
Milwaukee, Wisconsin 53226. Tel.: 414-456-4655; Fax: 414-456-6214; E-mail: seethara@mcw.edu.
Published, JBC Papers in Press, October 1, 2001, DOI 10.1074/jbc.M106419200
2
R. Yammani, S. Seetharam, and B. Seetharam,
unpublished observations.
The abbreviations used are:
Cbl, cobalamin
(vitamin B12);
EGF, epidermal growth factor;
IF, intrinsic
factor;
IFCR, intrinsic factor-cobalamin receptor;
PCR, polymerase
chain reaction;
bp, base pairs;
PAGE, polyacrylamide gel
electrophoresis.
Identification and Characterization of Two Distinct Ligand
Binding Regions of Cubilin*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
light chains (160 and 12 mM) (12, 13). The presence of
low and high affinity binding sites for high density lipoprotein and
light chain suggested that there are at least two binding sites for these ligands.
-barrel. The acronym CUB is derived from proteins
complement Clr/Cls, Uegf, and bone
morphogenic protein-1 that have these domains. Upstream of these CUB
domains, the rest of the cubilin molecule contains a 113-residue
N-terminal region followed by eight EGF-like repeats. Consistent with
these structural observations, the new name of cubilin will be used instead of IFCR throughout the rest of the manuscript.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor and, for
processing, mRNA encoding pre--lactamase provided by the
manufacturer were used as controls.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
[in a new window]
Fig. 1.
Schematic representation of various fragments
of rat cubilin generated by reverse transcriptase PCR. Other
details are provided under "Experimental
Procedures."
View larger version (122K):
[in a new window]
Fig. 2.
SDS-PAGE analysis of 35S-labeled
CUB domain fragments. In vitro transcribed and translated
cubilin fragments, CUB domains 1-4, 9-12, 12-17, and 18-27 were
separated on nonreducing SDS-PAGE before (lanes 1) and after
binding to and eluted from Sepharose-IF-Cbl (lanes 2) and
Sepharose-rat serum albumin columns (lanes 3). The bands
were visualized by fluorography.
View larger version (53K):
[in a new window]
Fig. 3.
SDS-PAGE analysis of 35S-labeled
N-terminal cubilin fragments. In vitro transcribed and
translated cubilin fragments, 113-residue N terminus + eight EGF-like + domains CUB 1 and 2 (lane 1) or the 113-residue N terminus
alone (lane 2) (A) or the translated products
bound to and eluted from Sepharose-IF-Cbl (lanes 1 and
2) (B) or Sepharose-rat serum albumin columns
(lanes 1 and 2) (C) were subjected to
nonreducing SDS-PAGE, and the bands were visualized by fluorography.
The position of the molecular weight markers (lane 0)
(A) are shown.
View larger version (65K):
[in a new window]
Fig. 4.
SDS-PAGE analysis of 35S-labeled
CUB domain (5-8) fragments. The indicated CUB domains were
translated (A), or the same fractions purified from
Sepharose-IF-Cbl (B) or Sepharose-rat serum albumin columns
(C) were subjected to nonreducing SDS-PAGE, and the bands
were visualized by fluorography. The position of molecular weight
markers (lane 0) and products of endogenous translation
(lane 1) are shown.
-factor was processed with core
N-glycosylation (Fig. 5A, compare mobilities of
lanes 5 and 6) and that Escherichia
coli -lactamase is processed with cleavage of
its signal peptide (Fig. 5A, lanes 7 and
8). These observations indicated that the lack of core
N-glycosylation of the cubilin fragments was not due to the
use of an inactive sample of canine pancreatic microsomes.
View larger version (83K):
[in a new window]
Fig. 5.
SDS-PAGE analysis of 35S-labeled
cubilin fragments translated in the presence of canine pancreatic
microsomal membranes. CUB domains 6-8 or the 113-residue N
terminus + EGF-like repeats + CUB domains 1 and 2 were translated in
the absence (A, lanes 1 and 3) and
presence (A, lanes 2 and 4) of canine
pancreatic microsomes, respectively, and translated products were
subjected to SDS-PAGE. As a positive control for microsomal activity,
mRNA encoding S. cerevisiae -factor was translated in
the absence (A, lane 5) and presence
(A, lane 6) of canine pancreatic microsomes.
E. coli -lactamase was used as a control for signal
peptide cleavage in the absence (A, lane 7) and
presence (A, lane 8) of canine pancreatic
microsomes. The 35S-labeled proteins synthesized, CUB 6-8
in the absence (B, lane 1) and presence
(B, lane 2) of canine pancreatic microsomes, and
the N-terminal cubilin fraction synthesized in the absence
(C, lane 1) and presence (C,
lane 2) of canine pancreatic microsomes were purified from
Sepharose IF-Cbl affinity columns and subjected to SDS-PAGE.
View larger version (33K):
[in a new window]
Fig. 6.
Ca2+-dependent ligand
binding of cubilin fragments. The 35S-labeled
113-residue N terminus or the CUB domains 7, 8, 7-8, and 6-8 were
purified by affinity chromatography in the presence of 10 mM CaCl2 or 10 mM EDTA and
separated by nonreducing SDS-PAGE. The bands visualized by fluorography
were quantified using the AMBIS radioimaging system. The dotted
horizontal line represents the total ligand binding in the
presence of 10 mM CaCl2 obtained using the
indicated cubilin fractions or the native membranes (50 µg of rat
renal apical brush border membrane protein) and is taken to represent
100%. The ratio of image density obtained for binding
(CaCl2/EDTA) is expressed as percentage of EDTA-inhibitable
binding. The values obtained are mean ± S.D. from triplicate
binding assays performed using four different translation
experiments.
View larger version (48K):
[in a new window]
Fig. 7.
Effect of cubilin antiserum on ligand
binding. The translated 35S-labeled 113-residue N
terminus or the indicated CUB domains were preincubated with
anti-cubilin antiserum (5 µl) for about 60 min at 22 °C. The
binding of these fractions to the ligand containing affinity matrix,
elution, and SDS-PAGE were carried out as before. The values reported
represent the percentage of ligand binding activity determined in the
presence of 10 mM CaCl2 and preimmune rabbit
serum (5 µl) and represent mean ± S.D. from four different
inhibition experiments from three separate translation reactions.
View larger version (108K):
[in a new window]
Fig. 8.
SDS-PAGE of EGF antiserum-treated,
affinity-purified N terminus cubilin fraction. Prior to
purification, the translated 35S-labeled 113-residue N
terminus + eight EGF-like repeats was treated with 5 µl of EGF
antiserum for 60 min at 22 °C and then subjected to ligand affinity
chromatography as indicated. The bound radioactivity was eluted and
subjected to SDS-PAGE, and the bands were visualized by fluorography.
The data shown is a typical representation of three separate SDS-PAGE
experiments from two separate competition experiments.
View larger version (40K):
[in a new window]
Fig. 9.
Competitive ligand binding. The
35S-labeled 113-residue N terminus and the CUB 6-8
fractions were preincubated for 60 min at 22 °C with rat IF-Cbl (50 ng) or albumin (100 ng) prior to their binding to Sepharose linked to
albumin or IF-Cbl, respectively. Data shown represents the mean ± S.D. of three different experiments.
View larger version (39K):
[in a new window]
Fig. 10.
Amino acid sequence alignment of functional
cubilin regions. Amino acid sequencing of the 113-residue N
terminus and CUB domains 7 and 8 was carried out by Jellyfish version
1.4 using matrix-Gonnet. Identical residues in all three regions or
between CUB domains 7 and 8 are indicated by vertical lines
and between CUB domain 7 or 8 and the N terminus by vertical
boxes. Potential sites for N-glycosylation are
indicated in bold letters and underlined. The
position of a proline residue implicated in high affinity IF-Cbl
binding to CUB 8 and its relative position in CUB domain 7 and the
113-residue N terminus are indicated by a star. Conserved
cysteine residues in CUB domains 7 and 8 are in dark boxes.
Proline residues that are conserved in all three regions elsewhere are
indicated in bold letters.
View larger version (82K):
[in a new window]
Fig. 11.
SDS-PAGE of 35S-labeled
113-residue N terminus and the CUB 6-8 fractions following reductive
alkylation. The nonreduced (lanes 1) or reduced and
alkylated (lanes 2) translated cubilin fragments or the
translated cubilin fragments purified from either Sepharose-IF-Cbl,
nonreduced (lanes 3) or reduced (lanes 4), or
Sepharose-rat albumin, nonreduced (lanes 5) or reduced
(lanes 6), were subjected to SDS-PAGE. The bands were
visualized by fluorography.
View larger version (15K):
[in a new window]
Fig. 12.
Reductive alkylation of CUB domains 5, 6, 7, and 8. The indicated translated 35S-labeled CUB
domains were reduced, alkylated, and subjected to SDS-PAGE. Lane
1, unreduced; lane 2, reduced. Details are provided
under "Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helixes (17) that are amphipathic in nature (14) and
are thought to interact with the lipid bilayer. The amphipathic nature
of the 113-residue N terminus is evident as it formed aggregates
following its synthesis (Fig. 3), and all the aggregated protein also
bound ligand. Moreover, when this region along with the EGF-like
repeats was stably expressed in Chinese hamster ovary cells, it was
retained within the cell unlike other fragments of cubilin, which were
all secreted efficiently. However, the ligand binding ability of this
fraction was not studied (14). Thus, based on the ability of this
fragment to bind the ligands and its hydrophobic nature, the N-terminal
region of cubilin may interact with the outer leaflet of the apical
bilayer membrane. Recently it has been shown (18) that the cubilin
fragment containing both the N terminus and the EGF-like repeats along
with CUB domains 1 and 2 bind to megalin in a
Ca2+-dependent manner. Moreover, this fragment
when reconstituted into egg phosphatidylcholine vesicles containing 10 mol % cholesterol in the presence of megalin demonstrated decreased
(75%) binding to these lipid
vesicles.2 Taken together,
these observations strongly suggest that megalin binding to the
N-terminal region may displace this region to the outer leaflet of the
bilayer to allow its ligand binding site to be exposed.
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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