Alternative Splicing in the Ligand Binding Domain of Mouse
ApoE Receptor-2 Produces Receptor Variants Binding Reelin but Not
2-Macroglobulin*
Christian
Brandes
,
Larissa
Kahr
,
Walter
Stockinger
,
Thomas
Hiesberger§,
Wolfgang J.
Schneider
, and
Johannes
Nimpf
¶
From the
Institute of Medical Biochemistry,
Department of Molecular Genetics, Biocenter and University of Vienna,
A 1030 Vienna, Austria and the § Department of Molecular
Genetics, University of Texas Southwestern Medical Center,
Dallas, Texas 75390-9046
Received for publication, March 26, 2001, and in revised form, April 4, 2001
 |
ABSTRACT |
LR7/8B and ApoER2 are recently discovered members
of the low density lipoprotein (LDL) receptor family. Although
structurally different, these two proteins are derived from homologous
genes in chicken and man by alternative splicing and contain 7 or 8 LDL
receptor ligand-binding repeats. Here we present the cDNA for
ApoER2 cloned from mouse brain and describe splice variants in the
ligand binding domain of this protein, which are distinct from those
present in man and chicken. The cloned cDNA is coding for a
receptor with only five LDL receptor ligand-binding repeats, i.e. comprising repeats 1-3, 7, and 8. Reverse
transcriptase-polymerase chain reaction analysis of mRNA from
murine brain revealed the existence of two additional transcripts. One
is lacking repeat 8, and in the other repeat 8 is substituted for by a
13-amino acid insertion with a consensus site for furin cleavage
arising from an additional small exon present in the murine gene. None of the transcripts in the mouse, however, contain repeats 4-6. In
murine placenta only the form containing repeats 1-3 and 7 and the
furin cleavage site is detectable. Analysis of the corresponding region
of the murine gene showed the existence of 6 exons coding for a total
of 8 ligand binding repeats, with one exon encoding repeats 4-6. Exon
trapping experiments demonstrated that this exon is constitutively
spliced out in all murine transcripts. Thus, the murine
ApoER2 gene codes for receptor variants harboring either 4 or 5 binding repeats only. Recombinant expression of the 5-repeat and
4-repeat variants showed that repeats 1-3, 7, and 8 are sufficient for
binding of
-very low density lipoprotein and reelin, but not for
recognition of
2-macroglobulin, which binds to the avian
homologue of ApoER2 harboring 8 ligand binding repeats.
 |
INTRODUCTION |
The low density lipoprotein receptor
(LDLR)1 family consists of a
growing number of structurally related composite cell surface receptors
with partially overlapping ligand specificity (1, 2). For example, the
LDLR harbors structurally and functionally defined modules,
corresponding to distinct exons in the gene (3). These modules are as
follows: (i) the "type A-binding repeats" (LA repeats) of ~40
residues each, displaying a triple disulfide bond-stabilized and
negatively charged surface mediating receptor/ligand interactions; (ii)
"type B repeats" (EG repeats), also containing six cysteines each;
EG repeats are homologous to regions in the epidermal growth factor
precursor; (iii) modules of ~50 residues with a consensus
tetrapeptide, Tyr-Trp-Thr-Asp (YWTD); (iv) a so-called
"O-linked sugar domain"; (v) a short transmembrane
domain of ~20 amino acids; and (vi) the cytoplasmic region with a
signal for receptor internalization via coated pits. LR7/8B (4, 5) and
its human homologue called ApoER2 (6, 7) belong to the close relatives
of the LDLR made up of exactly the same domains in the same order as in
the LDLR. The occurrence of distinct splice variants adds yet another
level of complexity to this family of proteins. LR7/8B expression in
chicken is highly restricted to the brain, where the protein resides in
large neurons and Purkinje cells, and in cells constituting brain
barrier systems such as the epithelial cells of the choroid plexus and
the arachnoidea and the endothelium of blood vessels (8). The finding
that chicken LR8B acts as receptor for
2-macroglobulin
(8) suggests a role in the clearance of
2-macroglobulin-proteinase complexes from the
cerebrospinal fluid. Ligand binding studies with two splice variants of
human ApoER2 demonstrated high affinity of the receptor to
-VLDL,
indicating that in mammals the receptor might be involved in
apoE-mediated transport processes in the brain as well (7). This is an
interesting aspect because a genetic link between certain alleles of
the ApoE gene and the development of late onset
Alzheimer's disease has been established (9). However, no differences
in the splice variant patterns of ApoER2 between control and
Alzheimer's patients exist (10).
Targeted disruption of the apoER2 gene alone or in
combination with that for the VLDL receptor gene revealed a key
function of both receptors during embryonic brain development (11).
Absence of functional ApoER2 and VLDL receptor leads to an inversion of cortical layers and absence of cerebellar foliation. This phenotype is
indistinguishable from that seen in animals carrying either a mutation
in the reeler gene or in the disabled gene (for
review see Refs. 12-14). Both ApoER2 and VLDL receptor bind reelin,
which is secreted by Cajal-Retzius cells (15, 16), and do so apparently together with cadherin-related neuronal receptors (17) and/or
3
1 integrin (18), which may act as
co-receptors for reelin to transmit the signal into migrating neurons.
Upon reelin stimulation the intracellular adapter protein disabled-1
(Dab1), which interacts with the cytoplasmic domains of apoER2 and
VLDLR (19, 20), becomes tyrosine-phosphorylated (21, 22). These data
suggest that ApoER2 and VLDL receptor directly relate the extracellular reelin signal into a cellular response via Dab1, leading to the ultimate cell responses required for the correct positioning of newly
generated neurons during brain development.
In addition, interaction screens with apoER2 and other members of the
receptor family have resulted in the identification of many other
candidate proteins interacting with the cytoplasmic domains of the
respective receptors, suggesting that they may be part of an intricate
network of signaling pathways (23, 24).
Here we present the full-length cDNA and the partial
characterization of the mouse apoER2 gene and show that
differential splicing events in the ligand binding domain produce
functional receptor variants that are distinct from those expressed in
chicken and man. Splicing is tissue-specific and is regulated during
brain development.
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MATERIALS AND METHODS |
5'-RACE Cloning of the Complete cDNA for Mouse
ApoER2--
The 5'-end of the mouse cDNA for apoER2 was cloned by
5'-RACE using the MarathonTM cDNA amplification kit
(CLONTECH) according to the manufacturer's protocol. First and second strand cDNA was synthesized from 5 µg
of poly(A)+ RNA and the poly(A) synthesis primer provided
by the kit. 5'-RACE was done in two successive rounds of PCR
amplification, with the sense primer located in an adapter ligated to
the cDNA. The antisense primer was located in the known sequence
from a partial cDNA clone (4). The first round, which resulted in
334 bp of new sequence, was done using primer ASR1
(5'-CCGCTCCTGGTTGCACCGTTTGATG). The second round was performed with
primer ASR2 (5'-GTCCTCGTCGCTGTTGTCCG) and resulted in additional 174 bp
of new sequence exceeding the start codon.
cDNA Preparation and PCR Analysis--
Total RNA was
produced from 200 mg of frozen tissue (total brain and placenta from
mature female Balb/c mice, and total chicken brain from female
Brown Derco) using TriReagent (Molecular Research Center, Inc.).
Poly(A)+ RNA was prepared from 500 µg of total RNA using
the Micro-Fast Track mRNA Isolation Kit (Invitrogen) according to
the manufacturer's protocol. First strand cDNA synthesis was
performed with 1 µg of poly(A)+ RNA and random hexamer
primers using SuperScript Reverse Transcriptase (Life Technologies,
Inc.). One-tenth of the cDNA was used for subsequent PCR in the
presence of 1.5 mM MgCl2, 0.2 mM
dNTPs, 2 units of Taq DNA polymerase (PerkinElmer Life
Sciences), and 1 µM of the appropriate primers on a
GeneAmp 2400 (PerkinElmer Life Sciences). PCR conditions are as
follows: 5 min initial denaturation at 94 °C, 1 min denaturation at
94 °C; 1 min annealing (for specific annealing temperatures, see
primers), and 1 min extension at 72 °C for 40 cycles. The following
primers have been used (numbers of the primers refer to the
respective primers shown in Fig. 2: mouse 1, 5'-CGAGAATGAGTTCCAGTGTGG;
mouse 2, 5'-CGTGAAGATCAGAGATGGGC, 1/2 annealing at 60 °C; mouse 3, 5'-CTGCCGAGAAGTTAAGCTGC; mouse 4, 5'-CCGCTCCTGGTTGCACCGTTTGATG, 3/4
annealing at 58 °C; mouse 5, 5'-GAGTTCCAGTGCAGCAACCG; mouse 6, 5'-CCGCTGCGGCAGGTGAACTGG, 5/6 annealing at 60 °C; chicken 7, 5'-CCAAGCAAGTATGCCCAGC; chicken 8, 5'-TTGGACACAGCCTGCCTCG, 7/8
annealing at 57 °C. PCR products were analyzed by agarose gel
electrophoresis. Bands were isolated from the gel using the QIAEX II
gel extraction kit (Qiagen) and subcloned into the pCR2.1 vector
(Invitrogen). Several clones from each fragment were isolated, and
positive clones were identified by PCR analysis and sequenced.
Partial Characterization of the Mouse ApoER2 Gene--
A mouse
ES P1 library (Genome Systems, Inc.) was screened by PCR using
the following primer pair: A, 5'-TGTCTGCACAATAACGGCGGCTGTTC, and B,
5'-ACAGGTCTTCTGGTCCAAGAGCTGGAAG. PCR conditions are as follows: 2 min
initial denaturation at 94 °C, 1 min denaturation at 94 °C, 1 min
annealing at 56 °C, 1 min extension at 72 °C for 35 cycles. Using
1 µg of genomic DNA, a 105-bp amplicon derived from exon 9 (EG repeat
A) was produced under these conditions. P1 plasmid DNA from positive
clones was prepared from 150-ml cultures (Escherichia coli
strain NS 3529, Genome Systems, Inc.) using the plasmid MidiKit
(Qiagen) according to manufacturer's instructions including an
additional phenol extraction step before loading on the column. P1 DNA
was digested with BglII and shotgun-subcloned into the
BamHI site of pBluescriptII (sK+). Clones were screened by
PCR using two independent primer combinations (pair 1, 5'-GCGCGGACGGCGACTTCACC, 5'-GCCTTCGATTCGTCAGAGCC; pair 2, 5'-CCGCCGTCCACGCACTCGCC, 5'-CGAGAATGAGTTCCAGTGTGG) located within the
5'-part and the 3'-part of the ligand binding domain, respectively. The
resulting clones were G14AS13 and 8AS-3.5 (see Fig. 3A). The
rest of the gene covering the ligand binding domain of the receptor was
covered by PCR clones derived from the undigested P1 clone with the
following primer combinations (see Fig. 3A): 1, 5'-TGCAGCTTCAGCATCTCTCC and 5'-GTCCTCGTCGCTGTTGTCCG; 2, 5'-CCTTGGTGTGGAGATGCGATGAGG and 5'-AGGTGAGCATGGCGGCCTGCC; 3, 5'-GCGCGGACGGCGACTTCACC and 5'-GCAGCTTAACTTCTCGGCAGG; 4, 5'-GACGGAGAGAAGGACTGTGAGG and 5'-TGGTCCACTCAGTCAACCTGTCC; 5, 5'-AGTTCTATGAGAACTCATCCACC and 5'-GTGCTCATGACTTAGTGATGTGG; 6, 5'-AGTGGCGAGTGCGTGGACGGC and 5'-TGAGGTCAGTGCAGATGTGG. PCR
products were isolated from the gel using the QIAEX II gel extraction
kit (Qiagen), subcloned into the pCR2.1 vector (Invitrogen), and sequenced.
Exon Trapping Experiments--
For the exon trap experiment two
constructs were made. One contained the exon coding for repeats 4-6
including 89 bp of the upstream and 563 bp of the downstream intron,
respectively (corresponding to genomic clone 5, Fig. 3). For
this, entire clone 5 was cloned into filled in SalI sites of
the exon trap vector pet01 (MoBiTec GmbH, Göttingen) resulting in
pEx4-6. The second construct contained genomic clone 5 plus clone
G14A13B3 (see Fig. 3), thus harboring two exons, one for repeat 2 and
the other for repeats 4-6. This construct (pEx2, 4-6) was made by
cloning the entire genomic clone G14A13B3 into pEx4-6 upstream of clone
5 using XbaI and NotI sites in the polylinker of
pet01. 3 µg of DNA was transfected into 293 cells using Lipofectin
reagent (Life Technologies, Inc.), and cells were grown on a 90-mm
plate. 48 h after transfection, cells were harvested, and mRNA
was prepared using the Micro-fast track from Invitrogen. 1 µg of
mRNA was taken and reverse-transcribed using 1 µl of Superscript
(Life Technologies, Inc.) and the cDNA primer provided by the exon
trap kit. RT-PCR was performed using 1/10th of the cDNA reaction
using two primers from the exon trap kit (5'-primer 2 and 3'-primer 3)
located 5' and 3' of the insert of pet01, respectively. PCR products
were analyzed on a 1.5% agarose gel and sequenced.
Expression of Chicken LR8B and Mouse ApoER2
4-6 and
ApoER2
4-6,8--
Chicken LR8B and mouse apoER2
4-6 and
apoER2
4-6,8 were expressed in the human embryonic kidney cell line
293. The full-length cDNA of mouse apoER2
4-6 and apoER2
4-6,8
were assembled by joining a partial cDNA for the mouse protein (4)
with the appropriate PCR products derived from mouse brain cDNA
with primers 5RS1 (5'-GGAGCCCCGGGCCCGCTATGG) and 5RAS6
(5'-GCTCATCAATGAGGACCACC) via an internal BglII site. The
products of the PCR reaction with 5RS1, 5RAS6 containing repeats 1-3,
7, and 8 and repeats 1-3 and 7 were purified on a 1% agarose gel,
cloned into pCR2.1, and sequenced. The partial cDNA (see above) was
cloned into the eukaryotic expression vector pCI-Neo. In order to
produce the full-length cDNAs, fragments containing 4 repeats and 5 repeats were cut out from the pCR2.1 constructs via an internal
BglII and the XhoI site (present in the
polylinker of pCR2.1) and cloned into the pCI-Neo construct containing
the rest of the cDNA. The construct used for expression of chicken LR8B in 293 cells has been described (8). Transfection of the cells was
performed using Lipofectin Reagent (Life Technologies, Inc.) according
to the manufacturer's protocol. Stable transformants were selected by
the addition of 500 mg/liter G418 to the medium (Dulbecco's modified
Eagle's medium (Life Technologies, Inc.), 10% fetal calf serum, 584 mg/liter glutamine).
Preparation of Cell Extracts, Electrophoresis, and Western
Blotting--
Total cell extracts from 293 cells expressing LR8B,
ApoER2
4-6, or ApoER2
4-6,8 were prepared as described for chicken
embryo fibroblasts (25). Electrophoresis, transfer to nitrocellulose membranes, and Western blotting were performed as described previously (26). The polyclonal antibodies against the cytoplasmic domains of
chicken LR8B (8) and mouse ApoER2 (24) are described in the respective references.
Binding and Internalization of
VLDL and
2M* by
Receptor-expressing Cells--
Rabbit
VLDL was prepared from the
plasma of animals fed a 2% cholesterol, 10% corn oil diet for 3 weeks
(27) and was radiolabeled with 125I by the iodine
monochloride method as described earlier (25). Lipoprotein
concentration is expressed in terms of protein content that was
measured by a modified Lowry procedure as described previously (28).
2M was isolated from chicken plasma as described (29). Native
2M was radiolabeled using Iodo-Gen pre-coated
iodination tubes according to the manufacturer's recommendation
(Pierce, catalog number 28601) to specific activities of 300-400
cpm/ng. Labeled
2M-trypsin complexes
(
2M*) were generated as described (29). Complete
activation of labeled
2M by trypsin was monitored by
native gel electrophoresis using the Tris borate system described by
van Leuven (30). Recombinant human RAP was produced as a glutathione
S-transferase (GST) fusion protein using a pGEX 2T-derived (Amersham Pharmacia Biotech) expression plasmid in DH5
bacteria (31).
Internalization of 125I-
VLDL and
125I-
2M* by LR8B-, ApoER2
4-6-, and
ApoER2
4-6,8-expressing 293 cells was measured according to the
standard protocol for uptake of LDL (32). In brief, 293 cells were
cultured in poly-L-lysine-treated 3-cm dishes and grown to
a 70% confluency. After three washes with phosphate-buffered saline,
cells were incubated with Dulbecco's modified Eagle's medium
containing 2% bovine serum albumin, 584 mg/liter glutamine, and
125I-labeled ligand at the indicated concentrations for
3 h at 37 °C. After washing, the cells were lysed by addition
of 1 ml of 0.1 N NaOH and incubation at 23 °C for 10 min. The radioactivity in the lysate was determined with a
-counter
(COBRA II, Packard Instrument Co.), and the protein concentration was
determined. Kd values were determined by Scatchard
analysis and expressed in nM using a
Mr = 350.000 for
VLDL.
Reelin Binding Studies--
293 cells expressing ApoER2
4-6
and ApoER2
4-6,8 and mock-transfected cells were washed and
resuspended in phosphate buffer and incubated with conditioned media
from reelin-expressing 293T cells as described (15). In brief, 293T
cells (60-80% confluent) were transfected with 7 µg of the reelin
construct pCRL. The following day, the culture medium was replaced with
a serum-reduced medium (Opti-MEM). After 2 more days the conditioned
medium was collected and used for incubation of ApoER2-expressing
cells. For ApoER2 expression, 293 cells were transiently transfected
with 10 µg of ApoER2
4-6 and ApoER2
4-6,8 constructs. After
48 h the cells were harvested, washed once with 1 ml of
phosphate-buffered saline, and incubated with 1 ml of the reelin
supernatant at 4 °C for 4 h in the absence or presence of
GST-RAP (30 µg/ml), EDTA (20 mM), or
-VLDL (50 µg/ml). CaCl2 was added to a final concentration of 0.5 mM. The cells were washed 2× with 1.5 ml of
phosphate-buffered saline and lysed with 50 µl buffer containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 20 mM sodium fluoride, 1 mM sodium orthovanadate, and proteinase inhibitors. After
30 min lysis on ice, the cell extracts were centrifuged at 13,000 × g for 30 min. 6-10 µl of the supernatant was
loaded on a 7.5% gel. Transfer to nitrocellulose was done as described
previously (16). The blot was incubated for 3 h with G10 antibody
(provided by Dr. Andre Goffinet, Namur Med. School, Namur, Belgium) at
a concentration of 1:1000. Goat anti-mouse HRP was used as a
secondary antibody at a concentration of 1:10,000. The blots were
developed using an ECL kit.
Alternatively, reelin binding to both splice variants of the receptor
was tested by a cell-independent assay described by Hiesberger et
al. (16). For this assay, cDNAs coding for the ligand binding
domains of ApoER2
4-6 and ApoER2
4-6,8, respectively, were
amplified by PCR using the respective full-length cDNAs as templates and fused to the constant human IgG domain as described (16)
resulting in ApoER2
4-6-Fc and ApoER2
4-6,8-Fc. Recombinant fusion
proteins were expressed in 293 cells, and the secreted proteins (1 ml
of cell supernatant) were bound to protein-A Sepharose (30 µl slurry)
as described. The protein-A Sepharose was then incubated with 500 µl
of conditioned media from reelin-expressing 293T cells in the presence
of 30 µg/ml RAP-GST or 20 mM EDTA or medium for 4 h
at 4 °C. Detection of bound reelin was performed as described (16)
using G10 as primary antibody and goat anti-mouse IgG conjugated to HRP
as second antibody. The amounts of receptor-Fc fusion proteins present
on the blot were analyzed by Western blotting using an HRP-coupled
anti-V5 antibody (Invitrogen).
 |
RESULTS AND DISCUSSION |
When we reported the cDNA cloning of chicken LR8B, we also
presented the amino acid sequence derived from a partial cDNA for the corresponding murine homologue (4). The 5'-end of that cDNA
defined the carboxyl-terminal part of the last LA-binding repeat of the
mouse receptor. By using this sequence information for RT-PCR
experiments, we were able to assign this sequence to repeat number 7 of
a mouse variant of LR7/8B that lacks repeat 8 due to differential
splicing (5). By using two rounds of 5'-RACE, we now have obtained the
full-length cDNA for mouse ApoER2 (Fig.
1). As outlined below, the murine
receptor appears to be a highly heterogeneous family of proteins due to
multiple alternative splicing events. Therefore we refer to the murine
protein as apoER2 as originally suggested for the human protein by
Yamamoto and colleagues (6). According to the rule of Van Heijne (33), we assigned the cleavage site for the signal peptide to amino acids
28/29, producing a mature receptor in which the first LA repeat is 7 amino acids longer than that of human apoER2 and 8 amino acids longer
than that of chicken LR7/8B. Surprisingly, the murine cDNA codes
for a receptor harboring only 5 LA repeats. Upon computer-assisted
sequence alignment (Geneworks) with human apoER2 and chicken LR8B, it
became evident that repeats 4-6 are missing from this transcript. As
reported recently, such a variant does exist for human ApoER2 (7).
Furthermore, the cloned murine transcript contains the eighth repeat
but not a 13-amino acid insertion that harbors a furin consensus
cleavage site at its carboxyl-terminal end. Alternative transcripts
containing such an insertion exist in man and mice but not in chicken
(5).

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Fig. 1.
Nucleotide and deduced amino acid
sequence of the cDNA for murine ApoER2. Amino acid sequence
numbering starts with the first amino acid in the mature protein after
cleavage of the putative signal sequence. The sequence corresponding to
the signal peptide is boxed. Cysteine residues are
encircled. The transmembrane domain is
underlined. The cytoplasmic internalization signal sequence
is boxed.
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Taken together, these results suggest that the ligand binding domain of
murine apoER2 might be subject to multiple alternative splicing events
producing a highly heterogeneous population of receptors. To evaluate
this situation in mice in close detail, we characterized the splice
variants in brain and placenta, the major sites of ApoER2 expression in
the mouse by RT-PCR experiments using different sets of primers
flanking the region between repeat 7 and the 5'-end of the EG repeats
(Fig. 2, primers 1, 2) and flanking the
cluster of repeats 4-6 (Fig. 2, primers 3, 4),
respectively. As shown in Fig. 2a, in adult mouse brain 3 distinct variants are present. These transcripts contain in addition to
repeats 1-3 the following: (i) either both repeats 7 and 8 (505 bp),
(ii) only repeat 7 together with a 13-amino acid insertion defining a
consensus furin cleavage site (421 bp), or (iii) repeat 7 only (382 bp). In embryonic brain (day 10), however, the large variant containing
repeat 8 is completely missing. This variant appears at day 12 establishing a splice variant pattern indistinguishable from that seen
in the adult brain (Fig. 2b). In mouse placenta a single
amplicon with a size of 421 bp is present (Fig. 2a). Upon
sequencing, this band turned out to be the transcript coding for the
receptor variant containing the insertion with the furin site and
lacking repeat 8. If the suggested cleavage at this site does occur,
the corresponding placental protein could be a soluble receptor
fragment with possibly different functions from those of the
full-length variants present in the brain.

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Fig. 2.
Analysis of splice variants in the ligand
binding domain of mouse ApoER2 and chicken LR7/8B. mRNA from
chicken and mouse brain and mouse placenta was used for cDNA
synthesis with reverse transcriptase. The resulting cDNAs and DNA
prepared from a mouse P1 clone was used for PCR amplification with the
indicated primer combinations. Amplified products were separated on
1.5% agarose gels (a-c, mouse; d, chicken). The
positions of the primers used are indicated by arrows in the
schematic, highlighting the relevant structural features of the
hypothetical cDNA deduced from the murine ApoER2 gene.
Empty numbered boxes and shaded boxes represent
sequences coding for LA repeats and EG repeats A and
B, respectively.
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By using a primer pair flanking repeats 4-6 (primers 3 and 4), the
only amplicon obtained using mRNA either from adult brain or
placenta had a length of 197 bp (Fig. 2c). Upon sequencing, it became evident that the amplified cDNA was identical to the corresponding region of the cDNA shown in Fig. 1 lacking repeats 4-6. As control we performed the same analysis with a chicken-specific primer pair using chicken brain mRNA as template. As seen in Fig. 2d, the only band obtained has a length of 614 bp and is
derived from a transcript containing these 3 LA repeats. Since exactly these repeats are encoded for by a single exon in the human gene (7),
we tested whether the corresponding exon was lost from the mouse gene
or whether it is deleted from the transcripts by alternative splicing.
Thus, we characterized the 5'-part of the murine apoER2 gene
which codes for the ligand binding domain of the receptor. As depicted
in Fig. 3A, the region of
interest of the murine apoER2 gene was cloned by a
combination of fragments of a P1 clone and of PCR-derived clones
obtained by respective primers located in the indicated regions of the
gene.

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Fig. 3.
Organization of the 5'-region of the murine
ApoER2 gene. A, the organization of
the 5'-region of the murine ApoER2 gene is schematically
shown. Exons are drawn as lines and introns as
triangles. The intron numbers are shown on
top. The exons are labeled with the designation of
corresponding structural elements of the receptor. Below,
P1-derived clones and PCR-derived clones covering the region of the
gene are drawn as lines (combinations of numbers
and letters refer to the primers described under
"Materials and Methods"). The schematic and the clones are not
drawn to scale. B, exon/intron organization of the murine
ApoER2 gene. Intronic sequences are shown in lowercase
letters, and exon sequences are in uppercase letters.
The intron numbers refer to the numbers used in A. Consensus
sequences for the 5'-splice donor and 3'-splice acceptor sites flanking
exon 5 are underlined (see text). The corresponding human
sequences in introns 4 and 5 are shown below the respective
mouse sequences. C, nucleotide and deduced amino acid
sequence of the cDNA encoded by exon 5 of the murine
ApoER2 gene. Intronic sequences are shown in lowercase
letters. Cysteine residues are encircled. Portions of
codons interrupted by introns are underlined.
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The apoER2 gene is organized much in the same way as that
for human apoER2 (7). In particular, the exon encoding the putative LA
repeats 4-6 is indeed present in the murine gene; the corresponding cDNA is shown in Fig. 3C. By having obtained the
sequence coding for repeats 4-6 from genomic cloning, we were able to
design another control experiment for the presence of transcripts
containing this exon in brain and placenta, respectively. A primer
combination corresponding to sequences within this exon (Fig. 2,
primers 5 and 6) were used to amplify corresponding transcripts by
RT-PCR. As seen in Fig. 2b, the expected amplicon with a
size of 293 bp was only obtained using the P1 clone as template but not
with cDNA derived from either brain or placental mRNA. This
experiment clearly demonstrates that this exon is missing from
transcripts in mice. It has been demonstrated that mutations in
sequences near splice junctions can affect splicing efficiency (34).
Thus, the flanking exon-intron junctions of exon 5 were compared with the consensus sequences for such regions (35). Both splice sites match
the consensus in that the 5'-donor site contains the 5'-GT, which is
essentially invariant, and the 3'-acceptor site contains a CAG which
follows a pyrimidine-rich stretch at the 3'-end of the corresponding
intron (the respective sequences are underlined in Fig.
3B). Furthermore, the respective intron sequences are completely conserved between the murine and the human gene (Fig. 3B). From the organization of the gene and the sequences of
the respective exon/intron junctions, it is not obvious why this exon is spliced out constitutively from the mature transcripts in mice, but
only facultatively in man (7). However, in a report where alternate
splicing patterns of ApoER2 in normal and Alzheimer brains was
investigated, the corresponding exon was missing in all human brain
samples examined (10). To clarify the situation, we cloned a genomic
fragment containing the exon coding for repeat 4-6 and parts of the
upstream and downstream introns into the exon trap vector pet01
creating pEx4-6 (Fig. 4A). As
internal control we combined this genomic fragment with another
fragment harboring the exon coding for repeat 2 (pEx24-6). When
transfected into 293 cells (Fig. 4B), pEx4-6 produced a
transcript of 246 bp, indistinguishable from that produced by the
vector alone. Sequencing of both products confirmed these bands to be
derived from the intrinsic exons present in the vector. pEx2,4-6,
however, produced a transcript with a length of 369 bp containing a
hybrid cDNA made up of the exons present in the vector and the exon
coding for repeat 2. The exon for repeats 4-6 again was missing from the transcript, indicating that this sequence is not recognized as exon
in this system.

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Fig. 4.
Exon trapping analysis of the exon
corresponding to repeat 4-6. A, the organization of
the exon trap constructs used in the experiment is schematically shown.
pet01 is the original vector (MoBiTec) with a multiple cloning site
within an intron flanked by two functional exons. For pEx4-6, genomic
clone 5 was inserted into the multiple cloning site of pet01. pEx2,4-6
was created by inserting clone G14A13B3 5' of clone 5. B, 3 µg of DNA of pet01, pEx4-6, and pEx2,4-6 were transfected into 293 cells; after 48 h mRNA was reverse-transcribed, and the
transcripts were analyzed by PCR as described under "Materials and
Methods." PCR products derived from the respective transfection
experiments are shown.
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Another very interesting feature of the murine gene is the fact that
the short insertion of 13 amino acids specifying a consensus furin
cleavage site, present in some splice variants of ApoER2 (5), is
encoded by a separate small exon and is not inserted by the use of an
alternative 5'-splice donor site, as might be expected for such a small
insertion. The 39-bp exon is located between those for LA repeat 8 and
EG repeat A, respectively (Fig. 5).
Sequence details of this region are shown in Fig. 5. Since a homologous
insertion is also present in variants of human ApoER2 (5), we assume
that in the human gene a corresponding exon is present in intron 6 (numbering according to Ref. 7).

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Fig. 5.
Partial sequence of the region of the murine
ApoER2 gene coding for the furin cleavage site.
Nucleotide sequence and deduced amino acid sequences of splice variants
derived thereof are represented in relation to a schematic representing
structural elements of the murine ApoER2 protein. Localization of
introns are highlighted by filled rectangles. Intronic
sequences are shown in lowercase letters and exon sequences
in uppercase letters. The putative furin cleavage consensus
sequence is underlined. Empty numbered boxes and
shaded boxes represent LA repeats and ER repeats A,
respectively.
|
|
To determine if the constitutive loss of repeats 4-6 in the murine
receptor which leads to the expression of receptor variants with either
4 or 5 ligand binding repeats results in distinct ligand specificity,
we expressed these variants, ApoER2
4-6 and ApoER2
4-6,8, in 293 cells, measured their affinities for
VLDL and
2M
(
2M*), and compared the results with those obtained with the chicken variant containing the maximum of 8 ligand binding repeats
(LR8B). The inset in Fig.
6D represents a Western blot with membrane detergent extracts from 293 cells expressing murine ApoER2
4-6,8 (lane 1), ApoER2
4-6 (lane 2),
and chicken LR8B (lane 3), respectively. Double bands seen
for the murine proteins most likely result from differential
glycosylation in this cell system. To test for the ability of these
receptors to interact with ligands, we used a combined binding and
internalization assay (8). As demonstrated in Fig. 6, A-C,
all three receptor variants bind
VLDL with high affinity
(Kd value of 53 nM for ApoER2
4-6,8; Kd value of 13 nM for ApoER2
4-6; and
Kd value of 8 nM for LR8B). This is not
surprising, since a human variant of ApoER2 harboring only 3 binding
repeats has been already shown to bind
VLDL efficiently (7).
Apparently, the affinity for
VLDL slightly decreases with the
successive loss of binding repeats. However, it was very interesting to
note that neither mouse ApoER2
4-6,8 nor ApoER2
4-6 bind
2M* (Fig. 6, D and E), in sharp
contrast to LR8B which binds
2M* with high affinity
(Kd value of 129 nM) (Fig.
6F, and cf. (8)). The small amount of
VLDL and
2M* binding to mock-transfected 293 cells is most likely due to low expression of intrinsic LDLR-related protein by these cells
(8). From an evolutionary point of view, it is interesting that the
loss of 3 binding repeats by constitutive splicing of the corresponding
exon in mice compromises the ability of the receptor to bind
2M*. Apparently, a minimum of 8 ligand binding repeats
seems necessary for recognition of
2M*, as suggested by
the fact that the chicken homologue of the VLDLR, i.e. LR8, contains 8 ligand binding repeats and binds
2M* with
high affinity (8), whereas the LDLR harboring seven repeats does
not.

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Fig. 6.
Binding and internalization of
2M* and VLDL
in cells expressing ApoER2 4-6,
ApoER2 4-6,8, or LR8B. 293 cells
expressing ApoER2 4-6,8 (A and D), ApoER2 4-6
(B and E), or LR8B (C and
F) and 293-cells transfected with the empty plasmid were
incubated for 3 h at 37 °C with the indicated concentration of
125I-labeled VLDL (A-C) or
2M* (D-F). Cell associated activity
(surface-bound and internalized) was measured as described under
"Materials and Methods." Values were corrected for non-saturable
binding and uptake in the presence of a 40-fold molar excess of
unlabeled VLDL or human recombinant RAP. Values are averages of
duplicate determinations, and Scatchard analysis was performed to
determine the indicated Kd values. D,
inset, Western blot analysis of 293 cells expressing
ApoER2 4-6,8 (lane 1), ApoER2 4-6 (lane 2),
and LR8B (lane 3), respectively. Triton X-100 detergent
extracts (50 µg of protein/lane) from transfected 293 cells were
subjected to electrophoresis under reducing conditions on a 4.5-18%
SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose
membrane that was incubated with antibodies specific for the respective
proteins (20 µg/ml). Bound IgG was visualized with protein A-HRP (1 µg/ml) and a chemiluminescence system.
|
|
Since it has recently been shown that one of the murine variants
(ApoER2
4-6) acts as receptor for reelin (15, 16), and as
demonstrated here (Fig. 7, A
and B), expression of the 4-repeat and 5-repeat variants in
the mouse brain is highly regulated during embryonic development, we
determined and compared reelin binding to both receptor variants.
Reelin binding was measured with two independent approaches, one using
receptor-expressing cells (15) and the other using soluble receptor
constructs as described recently (16). As demonstrated in Fig.
7A, cells expressing either ApoER2
4-6,8 or ApoER2
4-6
bind equal amounts of reelin produced by transfected 293T cells.
Binding of reelin to both receptor variants is abolished by EDTA,
GST-RAP, and
VLDL. This result was confirmed by the second assay
showing that soluble ApoER2 receptor constructs harboring either 4 or 5 binding repeats bind reelin with equal affinities (Fig. 7B).
Again, binding is completely abolished by an excess of GST-RAP or by
EDTA.

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Fig. 7.
Reelin binding to ApoER2 variants.
A, 293 cells expressing ApoER2 4-6,8 or ApoER2 4-6 and
293 cells transfected with the empty plasmid were harvested and
incubated with 1 ml of conditioned medium from reelin producing 293T
cells as described under "Materials and Methods." Incubations were
carried out without any addition or in the presence of GST-RAP (30 µg/ml), EDTA (20 mM), or -VLDL (50 µg/ml). After
incubation the cells were extensively washed, and bound reelin was
detected by lysis of the cells and subsequent Western blot analysis
using a specific antibody against reelin (G10). B,
recombinant fusion proteins containing the ligand binding domains of
ApoER2 4-6,8, ApoER2 4-6, or LDL receptor and the constant domain
of human IgG from 1 ml of culture supernatants from 293 cells
expressing ApoER2 4-6-Fc, ApoER2 4-6,8-Fc, or LDLR-Fc were bound to
protein A-Sepharose. The protein A-Sepharose was then incubated with
conditioned media from reelin expressing 293T cells in the presence of
30 µg/ml RAP-GST, 20 mM EDTA, or medium. Bound reelin was
detected by Western blot analysis using G10 as primary antibody and
goat anti-mouse IgG conjugated to HRP as second antibody. To test for
comparable amounts of fusion proteins present in the assay, the filter
was stripped and re-probed with an anti-V5 antibody.
|
|
The complex situation is summarized in Fig.
8, which compiles all identified
transcripts exhibiting variations in the ligand binding domain of the
receptor in different species. In chicken, where the prevalent
expression of LR7/8B occurs in brain, only two distinct variants
harboring 7 or 8 repeats exist. As analyzed at the mRNA level, the
furin cleavage site, which exists in man and mouse, is obviously
absent. However, the corresponding region of the chicken gene has not
been characterized to date, and it may well be that this exon is
constitutively spliced out in chicken transcripts. Therefore, the
question whether the corresponding exon in mammals was gained during
evolution cannot be answered yet. The mouse, as documented here,
produces three major transcripts varying in the ligand binding domain.
There are two significant differences to the transcripts in chicken.
First, none of the transcripts found contained repeats 4-6, which are
present on a single exon (exon 5). Genomic analysis suggests that this
exon is constitutively deleted by splicing out in mice. Second, in mice
there is an additional small exon following that for LA repeat 8, giving rise to a variant harboring a furin consensus cleavage site at
the carboxyl-terminal end of the ligand binding domain. Most
interestingly, this variant is the only one detectable in placenta,
showing that some of the splice events are tissue-specific.

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Fig. 8.
Comparison of the organization of the
5'-region of the mouse and the human ApoER2 genes and
transcripts derived from differential splicing in man, mouse, and
chicken. Boxes in the schematic represent exons within
the respective genes and transcripts. Numbers in the
boxes refer to the numbers of the LA repeats of the
corresponding proteins. Introns are shown as inverted filled
triangles and are numbered consecutively; unknown elements are
indicated by ?. The exons coding for the insertion
containing the furin cleavage site are labeled with the consensus
sequence (RRPR and RHPR). Shaded boxes
represent EG repeats A. Previously published information is indicated
by the respective references.
|
|
The situation in man is not as clear yet. Obviously, none of the
characterized transcripts contain repeat 8, and analysis of the human
gene suggests that the corresponding exon might have been lost (6, 7).
However, this exon seems to be present in the human gene, but a
mutation in the 5'-splice donor site causes this exon to be
constitutively skipped (36). In analogy to the mouse gene, a
corresponding exon coding for the furin-cleavage site insertion (5) is
likely present in the corresponding region of the human gene. In
addition to the mouse variants described herein, a receptor transcript
containing only repeats 1-3 has been reported to be present in humans
(7). As mentioned above, the use of exon 5, coding for repeats 4-6,
could not be confirmed in a recent study on normal and Alzheimer brains
(10).
So far, it has been shown that all human splice variants are able to
bind rabbit
-VLDL with similar affinity (6, 7). Thus, it is not
surprising that both murine ApoER2
4-6 and ApoER2
4-6,8 bind
-VLDL with high affinity. However, the fact that mouse ApoER2 does
not bind
2M* is interesting from an evolutionary point
of view considering that the chicken homologue recognizes
2M*. From a structural point of view the lack of
2M* binding to mouse ApoER2 is not surprising, since the
2M*-receptors found so far harbor a minimum of 8 ligand
binding repeats clustered in one domain (LDLR-related protein, chicken
LR8, chicken LR8B). Apparently, ApoER2 has lost its ability to bind
2M* but has gained other features during evolution from
birds to mammals. These features include the potential to be produced
as a soluble receptor fragment, and the ability to interact with
certain intracellular adapter proteins like JIP-1 and JIP-2, mediated
by an insertion in the cytoplasmic tail of the receptor (24).
Furthermore, this study demonstrates for the first time that both
murine ApoER2 variants containing either 4 or 5 LA repeats bind reelin
equally well. By taking into account that the expression of the
variants is highly regulated during brain development, it might be
possible that other ligands exist in the brain which discriminate
between both ApoER2 variants modulating the reelin response or
mediating a reelin-independent signal.
If indeed the arrangement of LA repeats in LDLR relatives defines their
ligand binding specificity, the splicing events in the
lr7/8B and ApoER2 gene might result in a complex
functional pattern of its products in different species and organs.
 |
ACKNOWLEDGEMENTS |
We appreciate the technical assistance of
Harald Rumpler and Robert Wandl. We thank Dr. Andre Goffinet for the
gift of the anti-reelin antibody G10 and Dr. Tom Curran for the reelin
expression plasmid.
 |
FOOTNOTES |
*
This work was supported by the Austrian Science Foundation
Grants P13931-MOB and F606 (to J. N.) and P13940-MOB (to W. J. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ312058 and AJ312059.
¶
To whom correspondence should be addressed: Institute of
Medical Biochemistry, Dept. of Molecular Genetics, Biocenter and University of Vienna, Dr. Bohrgasse 9/II, A-1030 Vienna, Austria. Tel.:
43-1-4277-61808; Fax: 43-1-4277-9618; E-mail:
JNIMPF@mol.univie.ac.at.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M102662200
 |
ABBREVIATIONS |
The abbreviations used are:
LDLR, low density
lipoprotein receptor;
VLDLR, very LDLR;
apo, apolipoprotein;
LR7/8B, LDLR relative with 7 or 8 LA repeats;
ApoER2, apoE receptor 2;
RAP, receptor-associated protein;
2M,
2-macroglobulin;
PCR, polymerase chain reaction;
LA, type A binding repeats;
EG, type B repeats;
RT, reverse transcriptase;
bp, base pair;
RACE, rapid amplification of cDNA ends;
HRP, horseradish peroxidase;
GST, glutathione
S-transferase.
 |
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