From the Lipoprotein & Atherosclerosis Group,
Departments of Pathology & Laboratory Medicine and Biochemistry,
University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4E9, Canada,
the ¶ Department of Molecular Genetics, Biocenter and University
of Vienna, Vienna, Austria, and the
Laboratory of Molecular
Biology, NCI, Division of Cancer Biology, Diagnosis and Centers,
National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The low density lipoprotein
receptor-related protein (LRP) is responsible for the clearance of
several physiological ligands including a complex of proteinase and
2-macroglobulin (
2M) and for the
entrance of Pseudomonas exotoxin A (PEA) into cells. We have prepared expression plasmids for the full-length chicken LRP
(designated LRP100) and two intermediates encoding 25 and 67% of the
receptor (designated LRP25 and LRP67, respectively) using overlapping
cDNA fragments. LRP25 and LRP67 encode the N-terminal 22 and 64%,
respectively, of LRP100 plus the transmembrane and intracellular
domains. Transient transfection of these plasmids into COS-7 cells
yielded recombinant proteins of expected molecular mass and
immunoreactivity. However, LRP100 was incompletely processed into
-
(515-kDa) and
- (85-kDa) chains and was poorly transported from the
endoplasmic reticulum to the Golgi compartment. Stable transformants of
LRP100, LRP67, and LRP25 were generated in a mutant Chinese hamster
ovary cell line that lacked expression of endogenous LRP and was
resistant to PEA. All forms of recombinant LRP proteins were
transported from the endoplasmic reticulum to the Golgi apparatus in
Chinese hamster ovary cells as shown by their sensitivity to
endoglycosidase H and resistance to neuraminidase. Cell surface
iodination and subcellular fractionation studies indicated that all
three LRP variants were expressed on the plasma membrane. Furthermore,
expression of the three LRP variants restored, to various degrees,
sensitivity to PEA and the ability to degrade methylamine-activated
2M (
2M*). These data suggest that
deletion of large internal portions of LRP, including the processing
site, does not prevent transport of LRP to the plasma membrane, nor does it abolish the interaction of LRP with
2M* or PEA.
This LRP expression system may allow for the characterization of
domains within LRP responsible for its multifunctionality.
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INTRODUCTION |
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The low density lipoprotein receptor-related protein
(LRP)1 of chicken somatic
cells has been characterized (1). Comparison of amino acid sequences
between the chicken LRP (4522 amino acids) and the human counterpart
(4525 amino acids) (2) has revealed 83% sequence identity between the
two receptors. LRP is translated as a single polypeptide chain (600 kDa) and subsequently processed into - (515-kDa) and
- (85-kDa)
chains during its transport from the endoplasmic reticulum (ER) to the
cell surface (3). Processing of LRP is catalyzed by the
trans-Golgi endopeptidase furin (4) that recognizes the
RXRR consensus sequence (RHRR in human LRP and RNRR in
chicken LRP). The resulting
- and
-chains are noncovalently
associated on the cell surface (3). Intracellular transport of the
nascent LRP polypeptide from the ER to the Golgi apparatus requires a
39-kDa chaperone called the receptor-associated protein (RAP) (5, 6).
Recently, it has been suggested that RAP facilitates LRP folding in the
ER and prevents premature association of physiological ligands
concomitantly synthesized with the receptor (7, 8).
LRP is a type I membrane protein. Its extracellular -chain contains
31 class A ligand-binding motifs, arranged into four clusters (I
through IV), and 22 class B epidermal growth factor type repeats. The
-chain contains a single membrane-spanning segment followed by two
Asn-Pro-X-Tyr motifs at the carboxyl terminus (1, 2).
Probably because of the structural similarities between chicken and
human LRP, both receptors exhibit identical binding to a number of
diverse ligands, including lipid-associated apolipoprotein E (apoE),
activated
2-macroglobulin (
2M*),
vitellogenin, and RAP (1, 9). The structural basis for the
multifunctionality of ligand binding to LRP is not completely
understood. Analyses of recombinant LRP minireceptors (7, 11) and
proteolytic fragments (10) of human LRP have demonstrated that ligand
binding activity is associated mainly with class A motifs in clusters II and IV. Conversely, clusters I and III displayed only weak ligand
binding activity. Accumulating experimental evidence suggests that LRP
plays an important role in hepatic clearance of chylomicron remnants, a
process probably mediated by interplay between many factors including
apoE (13, 14), hepatic lipase (15), lipoprotein lipase (16), and
heparan sulfate proteoglycans (17), in addition to LRP. With the
recognition of the possible link between apoE metabolism and the
development of Alzheimer's disease, the potential role of LRP in the
pathophysiology of the central nervous system has also been suggested
(18).
LRP has also been suggested to serve as a receptor for Pseudomonas exotoxin A (PEA) (19, 21). PEA consists of three functional domains and exerts its toxicity by irreversibly inhibiting protein translation. Following internalization, PEA protein is cleaved by a cellular protease and translocated to the cytosol. The N-terminal domain of PEA mediates cell binding, while the central domain contains the translocating activity and acts as the substrate for proteolytic cleavage. The C-terminal domain of PEA possesses the enzymatic activity and catalyzes ADP-ribosylation of elongation factor 2, resulting in inhibition of protein synthesis and cell death (22, 23). The primary target of PEA is the liver (25, 26). A mutant Chinese hamster ovary (CHO) cell line has been isolated that lacks LRP and is resistant to the toxic effects of PEA (20).
In this study, we prepared expression plasmids for the full-length
chicken LRP and two deletion variants using LRP cDNA fragments and
expressed them in an LRP-deficient CHO cell line (LRP-null). We used
the cytotoxicity of PEA to monitor the function of the recombinant
receptor and found that expression of the full-length receptor in the
LRP-null cell line restored its sensitivity to the toxin. Furthermore,
the expressed chicken LRP also mediated the uptake and degradation of
2M*. This expression system will facilitate
investigations of the pathophysiology and cell biology of this enormous
cell surface receptor.
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EXPERIMENTAL PROCEDURES |
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Materials-- DNA restriction enzymes and endoglycosidase H (Endo H) were purchased from New England Biolabs. Neuraminidase was purchased from Boehringer Mannheim or New England Biolabs. All reagents for cell culture were purchased from Life Technologies, Inc. ProMixTM (a mix of [35S]methionine and [35S]cysteine; 1000 Ci/mmol), carrier-free Na125I, horseradish peroxidase-conjugated goat anti-rabbit IgG antibody, and the enhanced chemiluminescence (ECL) reagents for immunoblotting were obtained from Amersham Corp. Bicinchoninic acid protein assay reagent and D-Salt ExcelluloseTM columns were obtained from Pierce. An expression plasmid encoding human furin (pcDNA3hfurin) was a gift from N. Seidah (Clinical Research Institute of Montreal). The expression plasmid pcDNA3RAP that encodes human RAP (5) and a polyclonal antibody against human LRP (27) were gifts from G. Bu (Washington University, St. Louis, MO). Recombinant Pseudomonas exotoxin A was expressed in Escherichia coli and isolated from the periplasm according to procedures described previously (28).
Preparation of Expression Plasmids-- The expression plasmids pcLRP25, pcLRP67, and pcLRP100 were constructed by combining 12 chicken LRP cDNA fragments that span the total length of 15598 base pairs (Fig. 1A). Plasmids pcLRP25 and pcLRP67 encode the N-terminal 22 and 64%, of the full-length LRP, respectively, plus the transmembrane and intracellular domains. Inserts were cloned into the polylinker region of the pCMV5 vector (29) positioned between the cytomegalovirus promoter and enhancer sequences and the human growth hormone transcription termination and polyadenylation signals (Fig. 1B). A BamHI-EagI fragment (nucleotides 1229-4222), prepared from clones A5, E4, and N3 was ligated together with an EagI-BglII fragment (nucleotides 14524-15248, obtained from clone L-1) into the pCMV5 vector that had been digested with XbaI and BamHI to create pcLRP25. Next, an EagI-EagI fragment (nucleotides 4222-9865) was prepared from clones N1, Z19, and 18 and inserted into pcLRP25 that had been digested with EagI to produce pcLRP67. Finally, an XhoI-XhoI fragment that encoded the C-terminal portion of LRP (nucleotides 9112-15248) plus the hGH region of the pCMV5 vector was prepared by ligation of clones 28, N2, 2B1-2, 112, 2B4, and L-1 and inserted into pcLRP67 to create pcLRP100 encoding the full-length chicken LRP.
Cell Culture and Transfection-- COS-7 cells (100-mm) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Wild-type CHO-K1 and LRP-null CHO cell line (designated 13-5-1) were cultured in Ham's F-12 medium containing 10% fetal bovine serum. Transfection of pcLRP into COS-7 cells was achieved by calcium phosphate precipitation (30) using 10, 20, and 25 µg of pcLRP25, pcLRP67, and pcLRP100 plasmid DNA, respectively. In certain experiments, pcLRP100 (25 µg) was transfected together with plasmids encoding human furin (10 or 15 µg) or RAP (10 or 15 µg). Stable transfection into LRP-null CHO cells (100-mm) was achieved by cotransfection of 15 µg of pcLRP100, 10 µg of pcLRP25, or 20 µg of pcLRP67 with 0.15 µg of pSV2neo. Stable transformants were selected using 700 µM G418 and maintained with 500 µM G418.
Preparation of Cell Extract and Immunoblot
Analysis--
Transfected cells were washed with phosphate-buffered
saline (PBS), resuspended in buffer S (200 mM Tris-maleate,
pH 6.0, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 µM leupeptin, and 1.4% Triton X-100) and incubated on
ice for 30 min. Insoluble protein was removed by centrifugation (14,000 rpm, 40 min, 4 °C) in an Eppendorf microcentrifuge. The Triton
X-100-soluble cell proteins were collected from the supernatant,
incubated with an equal volume of buffer R (8 M urea, 2%
SDS, 10% glycerol, 10 mM Tris-HCl, pH 8.3, and 5%
-mercaptoethanol) at 70 °C for 15 min, and resolved by
electrophoresis on 3-8% gradient polyacrylamide gels containing 0.1%
SDS (SDS-PAGE). Proteins were electrophoretically transferred (6 h)
onto nitrocellulose membranes for immunoblot analysis. Rabbit antiserum
against the carboxyl-terminal 17 amino acids of the
-chain of
chicken LRP or the C-terminal 15 amino acids of the
-chain was used
as primary antibody (1) for ECL detection.
Fractionation of Subcellular Membranes-- Transfected CHO cells (ten 100-mm dishes) were washed and harvested into PBS. Cells were collected by low speed centrifugation and resuspended in 2 ml of buffer A (20 mM HEPES, pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.1 mM PMSF, 0.1 mM leupeptin, 40 µg/ml acetyl-leucyl-leucyl-norleucinal, 10 kallikrein-inactivating units/ml aprotinin) and homogenized by 20 passes through a ball bearing homogenizer (H and Y Enterprise, Redwood City, CA). After centrifugation of the homogenate (16,000 × g, 20 min), the supernatant was subjected to another centrifugation (250,000 × g, 1.5 h) in a TLA100.4 rotor to isolate microsomal membranes. The 16,000 × g pellet was resuspended in 1 ml of buffer B (20 mM HEPES, pH 7.4, 1 mM EDTA, 0.1 mM PMSF, 0.1 mM leupeptin, 40 µg/ml acetyl-leucyl-leucyl-norleucinal, 10 kallikrein-inactivating units/ml aprotinin) using a glass homogenizer (10 strokes of a Teflon pestle), layered on a 1.12 M sucrose cushion, and subjected to centrifugation (100,000 × g, 1 h) in an SW41 rotor. The plasma membrane fraction was collected from the interface of the sucrose cushion and pelleted by centrifugation (30,000 × g, 30 min) in a TLA100.4 rotor. The microsomal and plasma membranes were resuspended in buffer B.
Endoglycosidase H or Neuraminidase Digestion of LRP
Proteins--
For Endo H digestion, cell extracts (Triton
X-100-soluble proteins) or subcellular membrane fractions (in buffer B)
were mixed with SDS (final concentration of 0.5%) and
-mercaptoethanol (final concentration of 1%) and heated to
100 °C for 10 min. The sample was then acidified by the addition of
sodium citrate, pH 5.5, to a final concentration of 50 mM.
Endo H (500 units) was then added, and the mixture was incubated at
37 °C for 5 h. For neuraminidase experiments, cell extract or
membrane fraction was acidified by the addition of sodium citrate
buffer, pH 6.2, to a final concentration of 50 mM.
Neuraminidase (0.75 units) was added, and the mixture was incubated at
37 °C for 4 h. Both Endo H and neuraminidase reactions were
stopped by the addition of 100 µl of buffer R and resolved by
SDS-PAGE under reducing conditions. LRP was detected by immunoblotting
as described above.
Pseudomonas Exotoxin A Cytotoxicity Assay-- Cells were seeded (1 × 105 cells/well) in 24-well dishes and allowed to adhere for 24 h. PEA was added to the medium at indicated concentrations, and cells were incubated for up to 18 h. The cells were washed with PBS (37 °C) and labeled with [35S]methionine/cysteine (200 µCi/ml) for 1 h in a methionine- and cysteine-free medium. After labeling, cells were washed with cold PBS and lysed with 250 µl of lysis buffer (1 mM EDTA, 1% Triton X-100, 1% deoxycholic acid, 1% SDS, 1 mM dithiothreitol, 0.015% PMSF, 50 mM Tris-HCl, pH 8.0). Complete solubilization of cell protein was achieved by heating the samples at 75 °C for 30 min. Each lysate was then diluted 10-fold with water, and an aliquot was mixed with an equal volume of fetal bovine serum and spotted under gentle vacuum onto 23-mm nitrocellulose membranes (saturated with ice-cold 10% trichloroacetic acid) using a Millipore 1225 manifold system. Membranes were washed twice with ice-cold 10% trichloroacetic acid and once with water and then dried for liquid scintillation counting.
Cell Surface Iodination--
Transfected CHO cell monolayers
(80-90% confluent in 60-mm dishes) were washed twice, and the cells
were collected into 1 ml of PBS (pH 6.5). Na125I (300 µCi) and two IODO-BEADs (Pierce) were added, and the cell suspension
was mixed by rocking for 15 min at room temperature. The cell
suspension was separated from the IODO-BEAD, and the cells were
pelleted by centrifugation (3000 × g, 2 min).
Unincorporated radioiodine was removed by washing the cells three times
with PBS. The cell pellet was resuspended in 160 µl of buffer I (200 mM Tris-maleate, pH 6.0, 2 mM
CaCl2, 0.5 mM PMSF, 2.5 µM
leupeptin) and were lysed with 40 µl of 7% Triton X-100 at 4 °C
overnight. The lysate was then diluted to 0.5 ml with buffer I
containing 1.4% Triton X-100 and precleared with protein A-agarose
beads (40 µl of 50% suspension) for 2 h. After removing the
protein A beads, an aliquot of the supernatant was incubated with
anti--chain or anti-
-chain antibody (overnight at 4 °C) to
precipitate LRP. The immune complex was recovered with protein
A-agarose beads, washed extensively with buffer I containing 1.4%
Triton X-100, and eluted into buffer R. Following separation by
SDS-PAGE, the radiolabeled LRP was visualized by autoradiography.
Preparation of RAP-GST Fusion Protein--
The Salmonella
japonicum glutathione S-transferase (GST)/39-kDa
expression plasmid containing human 39-kDa protein (RAP) cDNA was
obtained from D. Strickland. The purification of GST/39-kDa protein
from E. coli (DH5) and of the 39-kDa protein after
thrombin cleavage were carried out essentially as described by Herz
et al. (31).
Purification, Iodination, and Uptake/Degradation of
2M*--
Human
2M was purified from
fresh plasma (obtained from the Blood Bank of the Ottawa Civic
Hospital) by Zn2+-chelate affinity chromatography as
described previously (32). Preparation of
2M* was
performed according to previously described procedures (12). The
purified
2M* (100 µg in 250 µl of PBS, pH 7.3) was
iodinated with one IODO-BEAD and 1 mCi of Na125I for 15 min. Unincorporated 125I was removed by passing the
reaction mixture over a D-Salt Excellulose column equilibrated with PBS
containing 0.1% bovine serum albumin. 125I-
2M* (8.9 × 103
cpm/ng) was collected in the void volume of the column effluent. LRP-null and LRP-transfected cells (confluent in 12-well dishes) were
incubated with 125I-
2M* (2.5 nM,
0.4 ml/well) in the presence or absence of RAP in F-12 medium
containing 5 mM CaCl2 and 6 mg/ml bovine serum albumin for up to 4 h at 4 °C for
2M* binding or
37 °C for
2M* degradation. For degradation, the cells
were then placed on ice, and the medium was collected into tubes
containing ice-cold trichloroacetic acid (final concentration, 20%).
After 30 min, trichloroacetic acid-insoluble material was pelleted by
centrifugation, and the supernatant was removed for determination of
trichloroacetic acid-soluble, non-iodide radioactivity as described
previously (33). For
2M* binding, the cells were washed
twice with cold PBS containing 6 mg/ml bovine serum albumin and twice
with cold PBS. The cells were then solubilized with 0.1 N
NaOH, and radioactivity was quantified.
Protein Assays-- Proteins were determined by the bicinchoninic acid method (Pierce) according to the manufacturer's instructions.
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RESULTS |
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Transient Expression of LRP in COS-7 Cells-- The expression plasmid encoding the full-length chicken LRP (LRP100) was prepared by combining 12 LRP cDNA fragments as indicated in Fig. 1A. Two intermediate plasmids, LRP25 and LRP67, representing ~25 and ~67%, respectively, of the full-length receptor, were also generated during the preparation of LRP100 (Fig. 1B). Fig. 2 shows immunoblot analysis of the LRPs treated with and without Endo H (Fig. 2A) or neuraminidase (Fig. 2B), and Fig. 2C represents the domain structures of LRP. LRP25 encodes the N-terminal 984 amino acids (including the 21-residue signal peptide, cluster I, and the first three class A repeats of cluster II) plus the C-terminal 125 amino acids (including the transmembrane domain and the intracellular domain) of LRP100. LRP67 contains the N-terminal 2865 amino acids (including clusters I and II and nine class A repeats of cluster III) plus the transmembrane and intracellular domains.
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Expression of LRP100 in CHO-K1 Cells--
We tested several
different cell lines and found that CHO-K1 cells were a suitable host
for functional expression of recombinant LRP. Stably transfected cells
expressing LRP100 were generated in a mutant CHO-K1 cell line that
lacked expression of endogenous LRP (Fig.
3, A and B,
LRP-null). This LRP-null cell line was resistant to PEA
toxicity and was unable to internalize activated 2M
(20). In LRP100-transfected cells, the majority of LRP protein was
processed into
- and
-chains that could be detected in both the
microsomes (Fig. 3A) and plasma membrane (Fig.
3B). The unprocessed LRP100 was found in the microsome
fraction but was not detectable in the plasma membrane (middle
two lanes in Fig. 3, A and B), indicating that the majority of the surface-presented LRP is proteolytically processed. Surface iodination experiments with intact
LRP100-transfected cells also demonstrated that the processed
-chain
was the predominant form presented on the cell surface, although a
small amount of unprocessed LRP was iodinated in these experiments
(data not shown). As expected, the LRP
-chain was sensitive to
neuraminidase (right two lanes in Fig. 3, A
and B). The LRP
-chain, like the native
-chain
(left two lanes in Fig. 2B), was also sensitive
to neuraminidase (middle two lanes in Fig. 3, A
and B), indicating that the LRP
-chain is sialylated.
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Functional Analysis of Recombinant LRP in Transfected Mutant CHO
Cells--
We tested if expression of LRP100 in LRP-null cells would
restore the toxicity of PEA. Preliminary time course experiments indicated that at 200 ng/ml, PEA exerted maximal inhibitory effect on
protein synthesis after a 12-h incubation (data not shown). The effect
of PEA dose was assessed in PEA toxicity assays using cells that had
been treated with PEA for 18 h (Fig.
4A). In wild-type CHO-K1
cells, incorporation of [35S]methionine/cysteine into
cell protein decreased with increasing PEA dose, while LRP-null cells
were insensitive to the toxin. In LRP100-transfected cells, expression
of the full-length LRP restored the toxicity of PEA to LRP-null cells.
The PEA dose required to reduce protein synthesis to 50% of untreated
cells (IC50) decreased from >500 ng/ml in LRP-null cells
(19) to 50 ng/ml in the LRP100-transfected cells (Fig. 4A).
The IC50 for wild-type CHO-K1 cells was approximately 25 ng/ml, similar to an earlier observation (19). When the apparent PEA
toxicity observed in wild-type CHO-K1 cells and in LRP100-transfected cells was corrected for the number of receptor molecules on the surface
(as determined by 125I-2M* binding at
4 °C), the chicken LRP gave a value that was two-thirds of the
endogenous LRP (Table I, sixth
column).
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Functional Analysis of Two Deletion LRP Variants--
We next
tested whether LRP25 and LRP67 could function as receptors for PEA and
2M*. In three LRP67-transfected cell lines, two bands
representing the sialylated LRP67 (mature form) and the asialyl LRP67
proteins were observed (Fig.
5A). The mature LRP67 was
resistant to Endo H digestion (Fig. 5B) and sensitive to
neuraminidase digestion (Fig. 5C) and was the major species (in comparison with the asialyl form) in the plasma membrane fraction (Fig. 5, B and C, bottom). Cell
surface presentation of the mature form of LRP67 was also demonstrated
by surface iodination experiment (data not shown). In contrast, the
asialyl form of LRP67 was found predominantly in the microsomal
membrane fraction (Fig. 5, B and C,
top) and was sensitive to Endo H digestion (Fig.
5B).
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DISCUSSION |
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In this study, the full-length chicken LRP was stably expressed in
a mutant CHO-K1 cell line that lacks endogenous LRP. Biochemical experiments, together with functional analyses, indicated that the
recombinant LRP protein was glycosylated, proteolytically processed
into - and
-chains, and presented on the cell surface as a
functional receptor. Thus, we have provided conclusive experimental evidence using expressed recombinant protein that LRP indeed serves as
a receptor for
2M (34) as well as confirmed that it
serves as a gate for receptor-mediated entrance of PEA into cells (18). In addition, analysis of two receptors with internal deletions has
revealed that at least part of the sequence elements responsible for
the binding of
2M, RAP, and PEA may be located within
the amino terminus of the receptor.
It has been proposed that LRP contains multiple binding sites for RAP. Existence of RAP-binding sites in clusters II and IV of LRP has been shown by studies using anchored minireceptors (11), soluble LRP fragments (35), or proteinase and CNBr digests of LRP (10). Using anchor-free, soluble LRP fragments that contained each of the four clusters of the class A ligand binding repeats, Bu and co-workers (7) have shown that RAP binds avidly to clusters II and IV and less avidly to cluster III but does not bind to cluster I. They have also shown that there are at least five independent RAP-binding sites within LRP (two in cluster II, one in cluster III, and two in cluster IV) and that RAP binding activity seems to be conferred primarily by the class A motifs (8). The current studies with the anchored minireceptor LRP25 (Fig. 8B) have provided new evidence that a RAP-binding site may reside within the first three class A repeats of cluster II.
The 2M binding site has been assigned to cluster II of
LRP, but the binding activity may not be conferred solely by the class A repeats. Studies with proteolytic fragments of LRP have suggested that some epidermal growth factor type repeats flanking cluster II of
class A motifs may also contribute to binding of
2M-proteinase complexes (10). A membrane-anchored
minireceptor that contained all eight class A repeats of cluster II but
not the neighboring fourth epidermal growth factor repeat did not show
binding to
2M (11). Our observations of binding and
degradation of
2M* by LRP25-transfected cells indicate
that structural determinants essential for binding of
2M* are encoded by the amino-terminal 22% of the LRP
molecule, a region that also contains sequence determinants for binding
of RAP.
This study is the first attempt to define sequence elements within LRP
that are required for the entrance of PEA. Evidence that at low
concentrations PEA might enter cells via LRP-mediated endocytosis
includes (i) binding of PEA to LRP -chain and inhibition of binding
by RAP (19) and (ii) resistance to PEA toxicity of cells lacking LRP
expression (20, 21). The LRP-null cell line (13-5-1) used in the
present study was initially selected for its increased (100-fold) PEA
resistance and inability to internalize the
2M-proteinase complex (20). The current transfection
experiments have confirmed that the receptor responsible for PEA
entrance certainly is the receptor for
2M, and they have
also demonstrated that deletion of a significant portion of the LRP
molecule including the processing site (in LRP67) does not impair the
ability of PEA to exert its toxicity. It is noted that even in cells
that express an extremely high level of LRP25 (Fig. 8A), the
toxicity of PEA is only restored slightly (<10%). Therefore, the
affinity of PEA for LRP may be a function of the receptor length. The
LRP expression system established in the current study should be a suitable model to further define the retrograde trafficking of PEA in
cells.
Among all members of the low density lipoprotein receptor gene family, LRP is unique in its post-translational processing into two subunits. Similar processing occurs in the insulin receptor. Defective processing of the insulin receptor is associated with impaired insulin binding (<10% of normal) in transfected mutant CHO cells (36) and in some insulin-resistant diabetes mellitus patients (37). However, the functional expression of the membrane-anchored LRP25 and LRP67 demonstrated in this study suggests that the proteolytic cleavage of LRP may not be essential for the receptor's function. Abolishing the LRP processing in the two deletion variants does not seem to adversely affect their post-translational modification (e.g. N-glycosylation and sialylation; although in the mutant cell lines screened there appear to be two glycosylated forms of LRP25 and LRP67), ER-to-Golgi transport, cell surface presentation, or endocytosis. Willnow et al. (11) have previously expressed a functional membrane-anchored minireceptor that contained cluster II plus the transmembrane and intracellular domains but lacked the processing site of LRP in ldlA7 cells. The minireceptor bound and degraded RAP and the tissue-type plasminogen activator/plasminogen activator inhibitor-1 complex (11). To date, the physiological significance of post-translational processing of LRP remains unknown.
An important finding of this study is that the chicken LRP cannot be
expressed on the cell surface in COS-7 cells. Biochemical analysis of
the carbohydrate moiety indicated that the recombinant LRP was retained
within the ER and was not processed into - and
-chains,
suggesting that the transport of the chicken receptor to the distal
Golgi was impaired. Previous transfection studies of human low density
lipoprotein receptor showed that the recombinant receptors could be
functionally expressed in COS cells (38, 39). The inability of COS
cells to transport recombinant LRP onto the cell surface may be
attributable to the failure of the cellular trafficking machinery to
correctly recognize the chicken receptor, since endogenous LRP was
shown to be fully processed into
- and
-chains and was presented
on the cell surface. Since the molecular chaperone RAP is essential for
LRP folding and also to prevent premature binding of the receptor to
its ligands (7, 8), we have considered the possibility that RAP might
be limiting in COS-7 cells. However, co-expression of human RAP with
LRP did not improve trafficking of the receptor. It is unlikely that
the lack of an effect of the human RAP expression on LRP trafficking is
the result of its inability to react with the chicken LRP, since our
in vitro studies demonstrated clearly that the human RAP can
effectively abolish uptake and degradation of
2M.
Currently, the reason for the inability of COS-7 cells to transport
recombinant LRP onto the cell surface is unexplained.
In summary, we have established an expression system for LRP, a multifunctional cell surface receptor involved in the catabolism of proteinases and lipid-associated proteins. The availability of an in vitro LRP expression system will assist in the identification of structural determinants that are responsible for the multiligand binding activity of LRP and will also facilitate investigations of the involvement of LRP in the development of premature atherosclerosis and Alzheimer's disease.
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ACKNOWLEDGEMENTS |
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We thank N. Seidah for the furin expression plasmid; G. Bu for the RAP expression plasmid and anti-LRP and anti-RAP antibodies; and D. Strickland for the GST/RAP expression plasmid. Work in J. N. laboratory is supported by the Austrian Science Foundation (Grant F-0606).
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FOOTNOTES |
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* This work was supported by Medical Research Council of Canada Grant MT-12931.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.
§ Supported by a studentship from the Medical Research Council of Canada.
** Research Scholar of the Heart & Stroke Foundation of Canada. To whom correspondence should be addressed. Tel.: 613-798-5555 (ext. 8711); Fax: 613-761-5281; E-mail: zyao{at}heartinst.on.ca.
1
The abbreviations used are: LRP, low density
lipoprotein receptor-related protein; ER, endoplasmic reticulum; RAP,
receptor-associated protein; apo, apolipoprotein; 2M,
2-macroglobulin; CHO, Chinese hamster ovary; PEA,
Pseudomonas exotoxin A;
2M*,
methylamine-activated
2M; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; PMSF,
phenylmethylsulfonyl fluoride; Endo H, endoglycosidase H; GST,
glutathione S-transferase.
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REFERENCES |
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