From the Departments of Pediatrics and Cell Biology
and Physiology, Washington University School of Medicine and St. Louis
Children's Hospital, St. Louis, Missouri 63110 and the
¶ Department of Biology, University of Chile,
Santiago, Chile
Received for publication, February 20, 2001
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ABSTRACT |
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The low density lipoprotein receptor (LDLR)
family is composed of a class of cell surface endocytic receptors that
recognize extracellular ligands and internalize them for degradation by lysosomes. In addition to LDLR, mammalian members of this family include the LDLR-related protein (LRP), the very low density
lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (apoER2),
and megalin. Herein we have analyzed the endocytic functions of the cytoplasmic tails of these receptors using LRP minireceptors, its
chimeric receptor constructs, and full-length VLDLR and apoER2 stably
expressed in LRP-null Chinese hamster ovary cells. We find that
the initial endocytosis rates mediated by different cytoplasmic tails
are significantly different, with half-times of ligand internalization ranging from less than 30 s to more than 8 min. The tail of LRP mediates the highest rate of endocytosis, whereas those of the VLDLR
and apoER2 exhibit least endocytosis function. Compared with the tail
of LRP, the tails of the LDLR and megalin display significantly lower
levels of endocytosis rates. Ligand degradation analyses strongly
support differential endocytosis rates initiated by these receptors.
Interestingly apoER2, which has recently been shown to mediate
intracellular signal transduction, exhibited the lowest level of ligand
degradation efficiency. These results thus suggest that the endocytic
functions of members of the LDLR family are distinct and that certain
receptors in this family may play their main roles in areas
other than receptor-mediated endocytosis.
The low density lipoprotein receptor
(LDLR)1 family includes five
members in mammals: the LDLR itself, the apolipoprotein E receptor 2 (apoER2), the very low density lipoprotein receptor (VLDLR), the
LDLR-related protein (LRP), and megalin (1-3). LDLR, VLDLR, and apoER2
have molecular masses of ~130 kDa, whereas LRP and megalin are
significantly larger with molecular masses of ~600 kDa. There are
several structural modules that are present in each member of the LDLR
family. These modules include 1) ligand-binding repeats of ~40 amino
acids that include six cysteine residues forming three disulfide bonds;
2) epidermal growth factor precursor repeats, which also contain six
cysteines residues each; and 3) modules of ~50 amino acids with a
consensus tetrapeptide, Tyr-Trp-Thr-Asp (YWTD). In addition to these
extracellular modules, each of these receptors also contains a single
transmembrane domain and a relatively short cytoplasmic tail with
potential endocytosis signals (1-5). A cluster of several
complement-type ligand binding repeats constitutes a ligand-binding
domain, and differential clustering of these repeats within a domain
may impart specificity with respect to ligand recognition (3). The
epidermal growth factor precursor homology domains and YWTD repeats are
necessary for the dissociation of ligands from the receptor in
endosomes (6, 7). Ligand interactions with all members of the LDLR
family can be antagonized by a receptor-associated protein (RAP), a
unique ligand frequently used as a tool in the study of ligand-receptor
interaction (8). RAP also functions intracellularly as a molecular
chaperone to facilitate receptor folding and trafficking within the
early secretory pathway (8).
Traditionally, all members of the LDLR family have been regarded as
cell surface endocytosis receptors that function in delivering their
ligands to lysosomes for degradation (1-3). However, recent studies
have revealed new roles for these receptors in signal transduction (9).
A set of cytoplasmic adaptor and scaffold proteins containing protein
interaction domain or PSD-95/DLG/Z0-1 domains have been shown to
bind to the cytoplasmic tails of members of the LDLR family (10-15).
These new findings suggest that members of the LDLR family may
participate in several signal transduction pathways including the
regulation of mitogen-activated protein kinases, cell adhesion,
vesicle trafficking, neurotransmission, and neuronal migration (9).
Cellular signaling through this class of receptors may be regulated by
receptor endocytosis (13). For example, binding of the adaptor protein
Dab1 (Disabled-1) to the
cytoplasmic domain of the LDLR impedes its interaction with the
endocytic machinery (13).
The relatively short cytoplasmic tails of LDLR, VLDLR, apoER2, LRP, and
megalin contain 50, 54, 115, 100, and 209 amino acid residues,
respectively (16-20). A common characteristic of the LDLR family
members is that at least one copy of the NPXY sequence is
found within their cytoplasmic tails. For LDLR, this NPXY
motif serves as a signal for receptor endocytosis through coated pits (4). However, we recently reported that the YXXL motif, but not the two NPXY sequences, within the cytoplasmic tail of
LRP serves as the dominant signal for receptor-mediated endocytosis (5). We also demonstrated that the distal dileucine motif and a serine
phosphorylation within the LRP tail contribute to receptor endocytosis
(5, 21). These results suggest that each member of the LDLR family may
utilize different potential signal(s) within their cytoplasmic tails
for receptor-mediated endocytosis.
In the present study, we have directly compared the endocytic functions
of the members of the LDLR family using LRP minireceptors, its chimeric
receptor constructs, and full-length VLDLR and apoER2 stably expressed
in LRP-null Chinese hamster ovary (CHO) cells. We find that the initial
endocytosis rates mediated by the cytoplasmic tails of the LDLR family
members differ significantly, suggesting that members of the LDLR
family may play their main roles in either receptor-mediated
endocytosis or signal transduction.
Materials--
Plasmid pLDLR-2 was obtained from American Type
Culture Collection. Plasmid pcDL-SR containing human VLDLR cDNA,
and plasmid pcDL-SR containing human apoER2 cDNA were kindly
provided by Dr. Tokuo Yamamoto (Tohoku University Gene Research Center,
Sendai, Japan). A human kidney cDNA library was obtained from
CLONTECH for polymerase chain reaction (PCR)
cloning of the megalin tail. Human recombinant RAP was expressed in a
glutathione S-transferase expression vector and isolated as
described previously (22). Human Constructs--
The construction of full-length human VLDLR with
an HA epitope near the amino terminus in pcDNA3 vector has been
described previously (27). Plasmid pcDL-SR containing human apoER2
cDNA was digested with XbaI and EcoRI, and
the full-length apoER2 cDNA was subcloned into pcDNA3.1( Cell Culture and Transfection--
LRP-null CHO cell line and
CHO-K1 (kindly provided by Dr. David FitzGerald, National Institutes of
Health; see Ref. 29) were cultured in Ham's F-12 medium containing
10% fetal bovine serum. Stable transfection into LRP-null CHO cells
was achieved by transfection of 30 µg of plasmid DNA in 10-cm dishes.
Stable transfectants were selected using 700 µg/ml G418 and
maintained with 400 µg/ml G418.
Metabolic Pulse-Chase Labeling and
Immunoprecipitation--
Metabolic labeling with
[35S]cysteine was performed essentially as described
before (22, 30). For pulse-chase experiments, cells were generally
pulse-labeled for 30 min with 200 µCi/ml [35S]cysteine
in cysteine-free medium and chased with serum-containing medium
for 0 or 120 min. Cells were lysed with 0.5 ml of lysis buffer
(phosphate-buffered saline containing 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride) at 4 °C for 30 min.
Immunoprecipitation was carried out essentially as described before
(28).
Western Blot Analysis--
Stably transfected CHO cells were
lysed with 0.5 ml of lysis buffer at 4 °C for 30 min. Equal
quantities of protein were subjected to SDS-PAGE (6%) under reducing
conditions. Following transfer to polyvinylidene difluoride membrane,
successive incubations with proper primary antibody and horseradish
peroxidase-conjugated secondary antibody were carried out for 60 min at
room temperature. The immunoreactive proteins were then detected using
the ECL system.
Kinetic Analysis of Endocytosis--
CHO cells were plated in
12-well plates at a density of 2 × 105 cells/well and
used after overnight culture (5). Cells were rinsed twice in ice-cold
ligand binding buffer (minimal Eagle's medium containing 0.6% bovine
serum albumin), and 125I-RAP was added at 5 nM
final concentration in cold ligand binding buffer (0.5 ml/well). The
binding of 125I-RAP was carried out at 4 °C for 30 min
with gentle rocking. Binding of 125I-RAP was specific,
i.e. the addition of 100-fold excess unlabeled RAP inhibited
binding by 90-95%. Unbound ligand was removed by washing cell
monolayers three times with cold binding buffer. Ice-cold stop/strip
solution (0.2 M acetic acid, pH 2.6, 0.1 M NaCl) was added to one set of plates without warming up and kept on
ice. The remaining plates were then placed in a 37 °C water bath,
and 0.5 ml of ligand binding buffer prewarmed to 37 °C was quickly
added to cell monolayers to initiate internalization. After each time
point, the plates were quickly placed on ice, and the ligand binding
buffer was replaced with cold stop/strip solution. Ligand that remained
on the cell surface was stripped by incubation of cell monolayers with
cold stop/strip solution for a total of 20 min (0.75 ml for 10 min,
twice) and counted. Cell monolayers were then solubilized with low SDS
lysis buffer and counted. The sum of ligand that was internalized plus
that remained on the cell surface after each assay was used as the maximum potential internalization. The fraction of internalized ligand
after each time point was calculated and plotted. Analyses of 125I-RAP Binding and
Degradation--
Cells (2 × 105) were seeded into
12-well dishes 1 day prior to assays. Assay buffer (minimal Eagle's
medium containing 0.6% bovine serum albumin with 5 nM
radioligand, 0.6 ml/well) was added to cell monolayers, in the absence
or the presence of 500 nm unlabeled RAP, followed with incubation for
1 h at 4 °C. Thereafter, overlying buffer containing unbound
ligand was removed, and cell monolayers were washed and lysed in low
SDS lysis buffer and counted.
125I-RAP degradation was measured using the methods
described (4, 5, 31). Briefly, 2 × 105 cells were
seeded into 12-well dishes 1 day prior to assays. Prewarmed assay
buffer with 5 nM 125I-RAP was added to cell
monolayers in the absence or the presence of unlabeled 500 nM RAP followed with incubation for 4 h at 37 °C.
Thereafter, the plates were quickly placed on ice. The medium overlying
the cell monolayers was collected, and proteins were precipitated by
addition of bovine serum albumin to 10 mg/ml and trichloroacetic acid
to 20%. Degradation of radioligand was defined as the appearance of
radioactive fragments in the overlying medium that were soluble in 20%
trichloroacetic acid. The cell monolayers were washed, and the ligand
that remained on the cell surface in the steady state was stripped by
incubation of cell monolayers with cold stop/strip solution and
counted. The protein concentration of each cell lysate was measured in
parallel dishes that did not contain LRP ligand. The degradation
efficiency was the value of the ratio of the degraded
125I-RAP divided by the cell surface bound
125I-RAP in the steady state and calculated relative to mLRP4T100.
Endocytosis Rates of Endogenous LRP and Its Minireceptor
mLRP4T100--
LRP is synthesized as a 600-kDa single chain precursor
that undergoes post-translational proteolytic processing within the trans-Golgi compartment, by the endopeptidase furin (32,
33). This post-translational processing results in the formation of mature LRP as a noncovalently associated heterodimer, consisting of the
extracellular 515-kDa chain and the transmembrane 85-kDa chain (32). We
generated an LRP minireceptor that mimics the function and trafficking
of LRP. This LRP minireceptor composed of residues 3274-4525 of the
full-length LRP (28), which includes the fourth cluster of ligand
binding repeats and the entire carboxyl terminus of the receptor
(designated mLRP4T100, with "m" representing membrane-containing,
"4" representing the fourth cluster of ligand binding repeats,
"T" representing cytoplasmic tail, and "100" representing the
100 amino acid residues within the LRP tail; see Fig.
1A and Ref. 28). To facilitate
immunodetection, an HA epitope was included near the amino terminus of
mLRP4T100.
Our previous studies have shown that the fourth ligand-binding domain
of LRP binds RAP with high affinity (25) and that mLRP4T100
internalizes RAP with high efficiency (5). To examine whether there is
a difference between the endogenous LRP and mLRP4T100 for ligand
internalization, we compared the endocytosis rates of these two
receptors. Because LRP is the only receptor that binds RAP with high
affinity in CHO-K1 cells (34), we utilized 125I-RAP for our
endocytosis assays. Fig. 2A
shows the endocytosis rates of endogenous LRP in CHO-K1 cells and
mLRP4T100 stably expressed in LRP-null CHO cells. The endocytosis rates
of LRP and mLRP4T100 were extremely fast with internalization
half-times of less than 30 s and were virtually indistinguishable
from one another.
To confirm this finding with a physiological ligand, we measured the
endocytosis rate of LRP in CHO-K1 cells using LRP unique ligand
VLDLR and apoER2 Exhibit Low Endocytosis Rates--
Using LRP-null
CHO cells, we generated stably transfected cell lines expressing human
VLDLR and human apoER2. To examine whether these two receptors are
expressed in the correct forms, we performed metabolic pulse-chase
labeling and immunoprecipitation. Thus, stably transfected CHO cells
were metabolically pulse-labeled with [35S]cysteine for
30 min and chased for 0 or 120 min with complete medium, followed by
immunoprecipitation with anti-HA antibody for VLDLR or polyclonal
anti-apoER2 antibody for apoER2 (Fig. 3A). For both VLDLR and
apoER2, only one band around 130 kDa is detected prior to the chase.
This band represents the full-length ER precursor form of VLDLR or
apoER2 that lacks complex sugar modification (Fig. 3A,
indicated by an arrow). After 120 min of chase, one
additional band representing receptor mature form (indicated by an
arrowhead) is seen. It is also noted that apoER2 has higher level of mature form than VLDLR does following the chase (Fig. 3A). At the steady state, the ER form and the mature form of
VLDLR are expressed at similar levels and are migrated closely to each other as analyzed by Western blotting (Fig. 3B). For apoER2,
the level of apoER2 ER form is very low, whereas the level of apoER2 mature form is relatively high. As expected, the apoER2 mature form
migrated significantly more slowly than its ER form (Fig. 3B). Taken together, these results indicate that VLDLR and
apoER2 are expressed in correct forms in LRP-null CHO cells.
We next compared the endocytosis rates of mLRP4T100 with VLDLR
and apoER2 stably expressed in LRP-null CHO cells. Surprisingly, the
endocytosis rates of VLDLR and apoER2 were extremely low (Fig. 3C), with half-times of RAP internalization over 8 min
(Table I). At 1 min, VLDLR and apoER2
internalized 9.2 and 10%, respectively, of the total cell-associated
125I-RAP, corresponding to 15-18% of mLRP4T100
endocytosis (Figs. 3C and 5B). These results
indicate that VLDLR and apoER2 exhibit extremely low endocytosis rates
compared with LRP.
LDLR Tail and Megalin Tail Display Reduced Endocytosis Rates
Compared with LRP Tail--
At present, the ligand binding regions of
extracellular domain of megalin are yet to be defined. The very large
size of megalin (~600 kDa) and the lack of full-length cDNA also
limits its molecular manipulation and expression via transfection. For
LDLR, RAP binding affinity to LDLR is significantly lower than that of
other LDLR family members. Thus, we made two chimeric receptors that
contain LRP fourth ligand-binding domain, and LDLR or megalin
cytoplasmic tail. To generate the chimeras, we replaced the LRP tail of
mLRP4T100 with cytoplasmic tails derived from LDLR and megalin (Fig.
1B). Using LRP-null CHO cells, we generated stably
transfected cell lines expressing mLRP4-LDLR and mLRP4-megalin.
To examine whether the chimeric receptors were expressed in the correct
forms when they were stably transfected in LRP-null CHO cells, we
performed metabolic pulse-chase labeling and immunoprecipitation with
anti-HA antibody. For mLRL4T100, only one band around 200 kDa is
detected prior to the chase. This band represents the full-length ER
precursor form that lacks complex sugar modification (Fig. 4A, indicated by
arrow). After 120 min of chase, two additional bands are
seen. The 85- and 120-kDa bands represent the furin-processed minireceptor forms that correspond to the LRP-85 (Fig. 4A,
indicated by an open arrowhead) and LRP-ligand-binding
domain 4 (Fig. 4A, indicated by a closed
arrowhead), respectively (28). mLRP4-LDLR and mLRP4-megalin
exhibited a similar banding pattern on SDS-PAGE. The 120-kDa
furin-processed forms are identical to that of mLRP4T100, whereas the
ER forms and the other processed forms migrate faster or slower than
that of mLRP4T100 depending upon the length of the cytoplasmic tail.
The two processed forms of mLRP4-megalin migrate at similar positions
on 6% SDS-PAGE. At the steady state levels, these three receptors
exhibit a similar expression pattern (Fig. 4B). For
mLRP4T100, two distinct bands are seen on 6% SDS-PAGE gel under
reducing conditions. Because the HA epitope is near the amino terminus
of the minireceptor, Western blot analyses with anti-HA antibody do not
detect the LRP-85 band. Taken together, these results indicate that
mLRP4-LDLR and mLRP4-megalin are properly expressed and processed in
LRP-null CHO cells.
We next compared the endocytosis rate of mLRP4T100 with that of the
chimeric receptors stably expressed in LRP-null CHO cells. As shown in
Fig. 4C, the endocytosis rates of mLRP4-LDLR and
mLRP4-megalin were similar. However, compared with mLRP4T100, the
endocytosis rates mediated by LDLR tail and megalin tail were
significantly low, with half-times of RAP internalization of 4.8 and
3.1 min, respectively (Fig. 4C and Table I). At 1 min,
mLRP4-LDLR and mLRP4-megalin internalized 14 and 18%, respectively, of
the total cell-associated 125I-RAP, corresponding to
22-30% of mLRP4T100 endocytosis (Figs. 4C and
5A). These results indicate
that the LDLR tail and the megalin tail display reduced endocytosis
compared with the LRP tail.
Comparison of Endocytosis Rate of mLRP4T100 Endocytosis Mutant with
That of mLRP4-LDLR, mLRP4-megalin, VLDLR, and apoER2--
To further
characterize the endocytosis rates of mLRP4-LDLR, mLRP4-megalin, VLDLR,
and apoER2, we compared these with that of mLRP4T100(Y63A), which is a
LRP minireceptor endocytosis mutant. This mutant contains a
substitution of an alanine for the tyrosine residue, which is within
the LRP dominant endocytosis motif YXXL (Fig.
1B). Our previous studies have demonstrated that the
endocytosis rate of mLRP4T100(Y63A) was significantly lower than that
of mLRP4T100 (5). Fig. 5 shows that the endocytosis rate of
mLRP4T100(Y63A) is slightly lower than that of mLRP4-megalin, similar
to that of mLRP4-LDLR, but is significantly higher than that of VLDLR and apoER2. These results confirm that the LDLR tail and the megalin tail display reduced endocytosis compared with the LRP tail, whereas VLDLR and apoER2 exhibit extremely low endocytosis rates.
Low Efficiency in Ligand Degradation by mLRP4-LDLR, mLRP4-megalin,
VLDLR, and apoER2--
Having established that there are significant
differences in the endocytic function among the tails of the LDLR
family members, we then investigated ligand degradation efficiency for
these receptors. To assess relative amounts of the receptors on the
cell surface, we analyzed the ligand binding activity of the receptors
stably expressed in CHO cells at 4 °C. As shown in Fig.
6, CHO cells expressing mLRP4T100
exhibited a moderate level of cell surface 125I-RAP
binding, whereas CHO cells transfected with the pcDNA3 vector alone
exhibited only ~10% of RAP binding seen with mLRP4T100. This minimal
amount of RAP binding to pcDNA3-transfected cells is likely
mediated by cell surface heparan sulfate proteoglycan (35). However,
CHO cells expressing mLRP4-LDLR, mLRP4-megalin, VLDLR, and apoER2
exhibited higher levels of cell surface RAP binding activity, which
correspond to 225, 299, 289, and 220% of mLRP4T100
125I-RAP binding activity, respectively (Fig. 6). Because
RAP has similar binding affinities to mLRP4T100, VLDLR, and apoER2 (21, 36, 37), we concluded that the cell surface level of mLRP4T100 is
significantly lower than that of mLRP4-LDLR, mLRP4-megalin, VLDLR, and
apoER2.
To quantify the efficiency of ligand degradation, we incubated stably
transfected cell lines with 5 nM 125I-RAP for
4 h at 37 °C, and the amount of 125I-RAP bound at
the surface in the steady state was determined by its susceptibility to
release with acid buffer (pH 2.6). We also measured the amount of
125I-RAP that had been degraded and released into the
medium. The degradation efficiency was the value of the ratio of the
degraded 125I-RAP divided by the cell bound
125I-RAP at the steady state and calculated relative to
mLRP4T100. As shown in Fig.
7A, after incubation with
125I-RAP at 37 °C for 4 h, the amounts of
125I-RAP bound at the cell surface at the steady state were
significantly different. CHO cells stably transfected with mLRP4T100
exhibited lowest level of 125I-RAP binding. Interestingly,
CHO cells stably transfected with apoER2 exhibited highest level of
125I-RAP binding, suggesting a large amount of
125I-RAP accumulated at the cell surface. Indeed, Fig.
7B shows that CHO cells stably transfected with apoER2
degraded a less amount of 125I-RAP. Following normalization
for the amount of 125I-RAP bound at the surface at the
steady state, 125I-RAP degradation efficiency mediated by
mLRP4-LDLR, mLRP4-megalin, VLDLR, and apoER2 was only 23, 48, 25, and
7%, respectively, when compared with that observed with mLRP4T100
(Fig. 7C).
All members of LDLR family are recognized as cell surface
endocytisis receptors. However, the endocytosis rates mediated by these
receptors are unclear. In the present study, we provide direct evidence
that there are distinct differences in endocytic functions among the
tails of the LDLR family members. We found that the tail of LRP
supports the most efficient endocytosis, whereas LDLR tail and megalin
tail display reduced endocytosis rates compared with LRP tail, while
VLDLR and apoER2 exhibit minimal endocytosis function.
It is not surprising that among all members of the LDLR family
examined, the LRP tail supports receptor endocytosis to the greatest
degree. LRP belongs to the class of receptors that undergo constitutive
endocytosis in the presence or absence of ligand. This feature may be
determined by the constant exposure of its endocytosis signals and is
highlighted by its cell surface distribution concentrated within
clathrin-coated pits (5, 38). The tail of LRP consists of 100 amino
acid residues and contains multiple potential endocytosis signals
including two NPXY, one YXXL, and two
dileucine motifs. Our recent studies indicate that the YXXL motif within the cytoplasmic tail of LRP serves as the dominant signal
for LRP endocytosis. Furthermore, the distal dileucine motif and a
serine phosphorylation in its cytoplasmic tail also contributes to the
endocytosis of LRP (21). At present, it has been reported that LRP can
bind and internalize over 20 structurally and functionally distinct
ligands. In this report, we have demonstrated that LRP possesses
extremely fast endocytosis rate, which is consistent with its major
function as a clearance receptor.
In the present study, we found that the endocytosis rates mediated by
mLRP4-LDLR, mLRP4-megalin, VLDLR, and apER2 are significantly lower
than that of mLRP4T100. One common characteristic of the LDLR family
members is that each contains at least one copy of the NPXY
sequence within the cytoplasmic tail. The tails of LDLR, VLDLR, and
apoER2 contains one copy of the NPXY sequence, whereas the
megalin tail contains two copies of the NPXY sequence. The NPXY motif in the LDLR has been shown to serve as a signal
for receptor endocytosis through coated pits (4). Thus, it is possible that the NPXY motifs within the tails of VLDLR, apoER2, and
megalin also function as their dominant endocytosis signals.
In contrast to other family members, we found that the tails of VLDLR
and apoER2 support limited endocytosis. Recently, it has been
demonstrated that VLDLR and apoER2 serve as obligate components in
Reelin/Dab1-mediated neuronal migration (10, 11, 39, 40). Mice that
lack the genes for both VLDLR and apoER2 demonstrate a neurological and
neuroanatomical phenotype that is indistinguishable from that seen in
animals deficient in either Reelin or Dab1 (11). Thus, signal
transduction is likely the main function of VLDLR and apoER2.
It is worthy of note that compared with other members of the LDLR
family, apoER2 exhibits extremely low level of ligand degradation efficiency. This observation is in agreement with previous report by
Sun and Soutar (37), who found that Recent studies have suggested new roles for LDLR family members as
transducers of extracellular signals (9). For example, several
cytoplasmic adaptor and scaffold proteins containing protein interacting domain or PSD-95/DLG/Z0-1 domains, including Dab1, Dab2,
FE65, JIP-1, JIP-2, PSD-95, CAPON, and SEMCAP-1, bind to the
cytoplasmic tails of members of the LDLR family (10-15). Thus, the
present studies extend our understanding of the differences in the
biological activities of members of the LDLR family. Rapid receptor
internalization mediated by receptors such as LRP may result in rapid
desensitization of signals initiated by receptor ligation, whereas
slower endocytosis mediated by the VLDLR and apoER2 may allow for more
sustained signal transduction upon ligand binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin
(
2M) was purified from plasma and activated with
methylamine (to yield receptor-binding form,
2M*) as
described before (23). Polyclonal rabbit anti-human apoER2 antibodies were kindly provided by Dr. James S. Owen (University College London,
London, UK; see Ref. 24) and Dr. Johannes Nimpf (University of Vienna,
Vienna, Austria). Monoclonal anti-HA antibody has been described before
(25). Peroxidase labeled anti-mouse antibody and ECL system were from
Amersham Pharmacia Biotech. All tissue culture media, serum, and
plasticware were from Life Technologies, Inc. Immobilon-P transfer
membrane was from Millipore. Rainbow molecular weight markers were from
Bio-Rad. Proteinase inhibitor mixture CompleteTM was from
Roche Molecular Biochemicals. [35S]Cysteine was obtained
from ICN. Carrier-free Na125I was purchased from
PerkinElmer Life Sciences. IODO-GEN was from Pierce. Proteins were
iodinated by using the IODO-GEN method as described previously (26).
)
vector. The construction of the membrane-containing minireceptor of LRP
(mLRP4T100; see Fig. 1) with an HA epitope near the amino terminus in
pcDNA3 vector has been described previously (28). To generate the
chimeras, we cloned a PCR fragment encoding the cytoplasmic tail of the
LDLR, or megalin into a "tail-less" mLRP4 construct. To create the
tail-less LRP4 construct, a unique restriction site XhoI was
introduced after the transmembrane domain. Plasmid pLDLR-2 was used as
the PCR template for LDLR tail, whereas a human kidney cDNA library was used as template for the megalin tail. Each of the 5' PCR primers
contains a XhoI site, and all the 3' primers include
XbaI sites. These PCR fragments were digested with
XhoI and XbaI and ligated into the tail-less
mLRP4 construct digested with the same enzymes. Thus, compared with
mLRP4T100, these chimeric receptors include two extra amino acids
(leucine and glutamic acid) immediately after the transmembrane domain.
All oligonucleotides were synthesized at Washington University School
of Medicine Protein Chemistry Laboratory. All DNA sequences generated
by PCR were verified by DNA sequencing.
2M* internalization was carried out in the same way of RAP internalization, except that the ligand binding buffer contained 5 mM
CaCl2.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
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Fig. 1.
Schematic representation of mLRP4T100 and the
chimeric receptors. A, mLRP4T100 is depicted in
comparison with the full-length LRP molecule. The four putative
ligand-binding domains are labeled with numerals I, II, III, and IV.
B, schematic representation of potential endocytosis signals
within the tails of mLRP4T100, its endocytosis mutant, and the chimeric
receptors mLRP4-LDLR and mLRP4-megalin.
View larger version (9K):
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Fig. 2.
Endocytosis rates of endogenous LRP and its
minireceptor mLRP4T100. A, endocytosis of
125I-RAP by endogenous LRP and mLRP4T100. CHO-K1 and
LRP-null CHO cells stably transfected with mLRP4T100 were incubated
with 5 nM 125I-RAP at 4 °C for 30 min and
then shifted to 37 °C for the indicated times. The amounts of ligand
internalized as the fraction of the total cell-associated ligand (the
sum of the internalized ligand plus the ligand remaining on the cell
surface at the end of the assay; see "Experimental Procedures" for
further explanation) are plotted against time. Values are the averages
of triple determinations with the S.E. indicated by error
bars. B, endocytosis of
125I- 2M* by endogenous LRP.
CHO-K1 cells were incubated with 0.5 nM
125I-
2M* at 4 °C for 2 h, and
125I-
2M* internalization was carried out for
indicated time points as described for 125I-RAP
internalization. These experiments are representatives of several such
experiments performed with similar data.
2M*. Similar to that seen with 125I-RAP
internalization, LRP exhibited fast internalization of
125I-
2M* with a half-time of less than
30 s (Fig. 2B).
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Fig. 3.
VLDLR and apoER2 exhibit extremely low
endocytosis rates. A, metabolic pulse-chase labeling
and immunoprecipitation for VLDLR and apoER2. LRP-null CHO cells stably
transfected with full-length VLDLR and apoER2 were pulse-labeled with
[35S]cysteine for 30 min and chased for 0 or 120 min.
After each chase, cells were lysed, immunoprecipitated with anti-HA
antibody for VLDLR and with a polyclonal rabbit antibody for apoER2,
and analyzed via 6% SDS gels under reducing conditions. The positions
of the ER forms and mature forms are indicated by arrows and
open arrowheads, respectively. B, Western
blotting analysis of VLDLR and apoER2 stably transfected in LRP-null
CHO cells. The positions of the ER forms and mature forms are indicated
by arrows and open arrowheads, respectively. C,
endocytosis of 125I-RAP by mLRP4T100, VLDLR, and apoER2.
125I-RAP internalization in LRP-null CHO cells stably
transfected with mLRP4T100, VLDLR, and apER2 was carried out for
indicated time points as described in the legend to Fig. 2A.
Values are the averages of triple determinations with the S.E.
indicated by error bars. These experiments are
representatives of several such experiments performed with similar
data.
Half-times of 125I-RAP internalization in LRP-null CHO cells
initiated by the tails of LDLR family members
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Fig. 4.
LDLR tail and megalin tail display reduced
endocytosis rates compared with LRP tail. A, metabolic
pulse-chase labeling and immunoprecipitation for mLRP4T100, mLRP4-LDLR,
and mLRP4-megalin. LRP-null CHO cells stably transfected with
mLRP4T100, mLRP4-LDLR, and mLRP4-megalin were metabolic pulse-chase
labeled and immunoprecipitated with anti-HA antibody as described in
the legend to Fig. 3B. The positions of the ER form, the
LRP-ligand-binding domain 4, and the processed forms including
transmembrane domain and cytoplasm tail are indicated by
arrows, closed arrowheads, and open
arrowheads, respectively. Note that the two processed forms of
mLRP4-megalin migrate at similar positions on 6% SDS-PAGE.
B, Western blotting analysis of mLRP4T100, mLRP4-LDLR, and
mLRP4-megalin stably transfected in LRP-null CHO cells. C,
endocytosis of 125I-RAP by mLRP4T100, mLRP4-LDLR, and
mLRP4-megalin. 125I-RAP internalization in LRP-null CHO
cells stably transfected with mLRP4T100, mLRP4-LDLR, and mLRP4-megalin
was carried out for indicated time points as described in the legend to
Fig. 2A. Values are the averages of triple determinations
with the S.E. indicated by error bars. These experiments are
representatives of several such experiments performed with similar
data.
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[in a new window]
Fig. 5.
Comparison of endocytosis rate of mLRP4T100
endocytosis mutant with those of VLDLR, apoER2, mLRP4-LDLR, and
mLRP4-megalin. 125I-RAP internalization in LRP-null
CHO cells stably transfected with mLRP4T100(Y63A), mLRP4-LDLR,
mLRP4-megalin, VLDLR, and apER2 was carried out for indicated time
points as described in the legend to Fig. 2A. Values are the
averages of triple determinations with the S.E. indicated by
error bars. A, endocytosis of
125I-RAP by mLRP4T100(Y63A), mLRP4-LDLR, and mLRP4-megalin.
B, endocytosis of 125I-RAP by mLRP4T100(Y63A),
VLDLR, and apER2. These experiments are representatives of several such
experiments performed with similar data.
View larger version (15K):
[in a new window]
Fig. 6.
Ligand binding activity of mLRP4T100, VLDLR,
apoER2, and the chimeric receptors stably transfected in LRP-null CHO
cells. Binding of 5 nM 125I-RAP to
LRP-null CHO cells stably transfected with pcDNA3 or various
receptors was carried out for 1 h at 4 °C in the absence or
presence of 500 nM unlabeled RAP. Values are the average
specific binding of triple determinations with the S.E. indicated by
error bars. This experiment is a representative of two such
experiments performed with similar data.
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[in a new window]
Fig. 7.
VLDLR, apoER2, and the chimeric receptors
exhibit lower RAP-degradation efficiency. LRP-null CHO cells
stably transfected with mLRP4T100, mLRP4-LDLR, mLRP4-megalin, VLDLR, or
apoER2 were incubated with 5 nM 125I-RAP at
37 °C for 4 h in the presence or absence of 500 nM
unlabeled RAP. Cell surface bound 125I-RAP (A)
and 125I-RAP-degradation (B) were determined as
described in "Experimental Procedures." C, the ligand
degradation efficiency was the value of the ratio of the degraded
125I-RAP divided by the cell surface bound
125I-RAP and calculated relative to mLRP4T100. All values
are the averages of triplicate determinations with the S.E. indicated
by error bars. These experiments are representatives of two
such experiments performed with similar data.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-VLDL was poorly degraded by
apoER2 or its spliced variants compared with LDLR. The mechanistic basis thereof is not presently known. apoER2 shows high homology to
both the LDLR and VLDLR, including the positions of the exon/intron boundaries of the genes (41). The most notable difference between the
tail of apoER2 and that of LDLR or VLDLR is the presence of an
additional 59 amino acid residues encoded by an additional exon (19).
This domain has been recently reported to interact with two members of
the JUK-interacting protein family, JIP-1 and JIP-2
(JUK-interacting protein
1 and 2). These molecules belong to a group of
mitogen-activated protein kinase scaffolding proteins (13, 14). The
interaction with JIPs is specific for apoER2, because neither LDLR nor
VLDLR bind (14). Taken together, these observations suggest apoER2 is a
unique cell surface receptor within the LDLR family with its main
function likely in mediating signal transduction.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Alan Schwartz for critical readings and suggestions on the manuscript. We also thank Tokuo Yamamoto (Tohoku University Gene Research Center) for providing the cDNAs of VLDLR and apoER2, James S. Owen (University College London, London) and Johannes Nimpf (University of Vienna, Austria) for providing the anti-apoER2 antibodies, and David FitzGerald (NIH) for providing the LRP-null CHO cell line.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants NS37525, HL59150, and DK56783 (to G. B.) and by Fondecyt Grant 1990600, (to M. P. M.).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.
§ Recipient of a postdoctoral fellowship from the Heartland Affiliate of the American Heart Association.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Dept. of Pediatrics, Washington University School of Medicine, CB 8208, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-286-2860; Fax:
314-286-2894; E-mail: bu@kids.wustl.edu.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M101589200
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ABBREVIATIONS |
---|
The abbreviations used are:
LDL, low density
lipoprotein;
LDLR, LDL receptor;
2M,
2-macroglobulin;
apoER2, apolipoprotein E receptor-2;
CHO, Chinese hamster ovary;
LRP, LDL receptor-related protein;
RAP, receptor-associated protein;
VLDL, very low density lipoprotein;
VLDLR, VLDL receptor;
PCR, polymerase chain reaction;
HA, hemagglutinin;
PAGE, polyacrylamide gel electrophoresis;
ER, endoplasmic reticulum..
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