From the Department of Vascular Biology and
Thrombosis Research, University of Vienna, Schwarzspanierstrasse 17, ¶ Department of Cardiology, Vienna General Hospital, University of
Vienna, Währingergürtel 18-20, and
St. Anna
Children's Hospital, Children's Cancer Research Institute,
Kinderspitalgasse 6, 1090 Vienna, Austria
Received for publication, December 29, 2000, and in revised form, January 9, 2001
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ABSTRACT |
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Novel members of the low
density lipoprotein receptor family were identified in human
endothelial and vascular smooth muscle cells utilizing a
homology-cloning strategy. Four novel mRNA transcripts could be
identified as isoforms of the apolipoprotein E receptor 2 (apoEr2): one
form lacking three ligand binding repeats (nucleotides 497-883) but
containing a novel ligand binding repeat adjacent to a unique
cysteine-rich domain preceding the epidermal growth factor precursor
domain of apoEr2, forms lacking the O-linked sugar domain,
and forms containing a 59-amino acid deletion within the cytoplasmic
tail. By fluorescence in situ hybridization for chromosome
mapping, we could confirm that the novel alternative forms of apoEr2
are splice variants of transcripts from a single copy gene on
chromosome 1p34. To analyze whether the different splice variants of
apoEr2 mRNA are expressed in a splice variant-specific pattern, we
concentrated on the central nervous system, where high expression of
apoEr2 has been described originally. By means of splice
variant-specific in situ hybridization, we could confirm that apoEr2 mRNA is abundantly expressed in brain tissue and, with
exception of the newly identified ligand binding domain, all mRNA
splice variants exhibited a similar expression pattern. The mRNA of
the newly identified ligand binding domain, however, was expressed in
brain only in cells of the vascular wall, confirming data from Northern
blotting, where the mRNA of the newly identified ligand binding
domain was found in several tissues but was absent in brain tissue.
Members of the low density lipoprotein receptor
(LDLR)1 family (1) are
multi-functional clearance receptors able to bind a large number of
ligands, thus regulating lipid metabolism, extracellular proteolysis
(2, 3), and growth factor/cytokine-dependent pathways (4).
Within cells of the vascular wall, known members of the LDLR gene
family include the LDLR (5), the LDLR-related protein (LRP) (6), and
the very low density lipoprotein receptor (VLDLR) (7). Apart from
overlapping ligand specificity (3), LDLR binds predominantly to plasma
lipoproteins (8), whereas VLDLR and LRP also bind components of the
fibrinolytic system such as tissue-type plasminogen activator and
urokinase-type plasminogen activator, especially in complex with their
specific inhibitor, plasminogen activator inhibitor type 1 (9-12).
Based upon the observation that LRP expression is absent in vascular
endothelial cells (EC) (13) and that VLDLR expression is restricted to
smooth muscle cells (SMC) and ECs in specific vascular compartments
(14), we speculated that additional members of the LDLR family might exist on ECs. For this purpose we designed a PCR-based
homology-cloning strategy utilizing conserved structural elements
within the LDLR family. Such elements are (i) the ligand binding
domains containing the characteristic amino acid motif Ser-Asp-Glu
(SDE) within the variable number of ligand binding repeats, (ii) the
epidermal growth factor (EGF) precursor homology domains containing
several Tyr-Trp-Thr-Asp (YWTD) consensus tetrapeptides, and (iii) the cytoplasmic region. Applying this cloning strategy, we have cloned novel members of the LDLR family and identified these as splice variants of apoEr2 (15). To elucidate possible functional implications of these splice variants of apoEr2, we studied the splice-specific mRNA expression pattern in the brain, where apoEr2 was reported to
be expressed abundantly and splice variant-specific expression was
already described (16). We found that the mRNA splice variant containing the newly identified ligand binding domain was expressed in
brain only in vascular cells, whereas all other mRNA splice variants were found to be expressed in neuronal tissue in a similar pattern.
Cloning and Sequencing--
Poly(A+) RNA was
extracted from human umbilical vein endothelial cells (HUVEC), human
foreskin microvascular endothelial cells (HSMEC), and human arterial
SMC, isolated, and cultured as described (17) using oligo(dT)-cellulose
(Amersham Pharmacia Biotech). First-strand cDNA was synthesized
from 1 µg of mRNA at 42 °C using avian myeloblastosis virus
reverse transcriptase (Roche Molecular Biochemicals) and the degenerate
oligonucleotide LDLRF31 (5'-GTGGTYTTCIIRTASACRGGRTTGTCAAAGTT-3' (Y = C + T, R = A + G, S = G + C), corresponding to nucleotides 2477-25092 of human LDLR,
2604-2636 of human VLDLR, 13967-13999 of human LRP, and 13920-13952
of rat gp330 (18) cDNAs). Samples were adjusted to PCR buffer
conditions in a total volume of 50 µl with 30 pmol of primer LDLRF31
and 30 pmol of LDLRF50 (5'-CCIGMIGSIMTIGCWGTKGAYTGG-3' (M = A + C,
W = A + T, K = G + T), reverse complementary to nucleotides 1439-1463 of LDLR, 1656-1680 of VLDLR, 9776-9800 of LRP, and
9942-9966 of gp330), employing 2 units of Taq DNA
polymerase (PerkinElmer Life Sciences) and 0.1 unit of Pfu
DNA polymerase (Stratagene, La Jolla, CA). Hot start PCR (Thermal
Cycler 2400, PerkinElmer Life Sciences) was performed for 30 cycles
under the following conditions: 40 s at 94 °C, 40 s at
62 °C, and 1 min at 68 °C. Amplified products were separated on
1% agarose gels and stained with ethidium bromide. Products were
purified using the QIAEX DNA gel extraction kit (Qiagen, Hilden,
Germany) and analyzed by DNA sequencing with a model 310 DNA sequencer
(Applied Biosystems Inc., Foster City, CA). In the course of the
subsequent cloning of the 5' sequence we used the degenerate sense
primer LDLRF51 (5'-GACTGCGSIGAYGGITCIGAYGAG-3') corresponding to the
amino acid motif SDE and the specific antisense primer 5 (5'-CTGAGATGGTCTTATTGCCCGAG-3', nucleotides 1494-1516). Amplification
was performed as above. The 5'- and 3'-ends were identified from HUVEC
and HSMEC mRNAs by a rapid amplification of cDNA ends protocol
using the Marathon kit (CLONTECH, Palo Alto, CA)
according to the manufacturer's protocol. For that purpose, the
antisense primer 15 (5'-CTGGAAGCCTGCTGGGCACGT-3', nucleotides
1033-1053) was employed for 5'-adaptor amplification, and the sense
primer was employed for C5 (5'-CTCAACAGTCACTGCCGCTGTTATC-3', nucleotides 2409-2433) for 3' adaptor amplification. Both strands of
PCR products, in each case from three independent amplifications, were
completely sequenced.
Northern Blot Analysis--
Six µg of mRNAs from cultured
HUVEC, HSMEC, or SMC were electrophoresed on a denaturing
formaldehyde-agarose gel (1%) and transferred to a nylon membrane
(Duralon UV, Stratagene). Blotting, prehybridization, and hybridization
were performed according to standard procedures (19). Blots were
hybridized utilizing 32P-labeled DNA probes corresponding
to nucleotides 1444-2558 of apoEr2906 (Fig. 2),
nucleotides 1439-2509 of LDLR, nucleotides 1656-2604 of VLDLR, and
rat glyceraldehyde-3-phosphate dehydrogenase. The probes were labeled
by random priming to a specific activity of 1 × 10 9 cpm/µg using [32P]dCTP (Amersham Pharmacia Biotech) and
the DNA-labeling kit (Roche Molecular Biochemicals). Prehybridization
was performed for 2 h at 57 °C and hybridization overnight at
57 °C. Blots were washed at a final stringency of 1× SSC (0.15 M NaCl and 0.015 M sodium citrate), 5%
SDS at 60 °C. Blots were exposed to autoradiography film (Amersham
Pharmacia Biotech).
Chromosomal Localization--
The chromosomal localization of
the transcripts was determined by fluorescence in situ
hybridization (20) using nucleotides 1444-2558 of
apoEr2906 as a probe. The probe was labeled with biotinylated-dUTP using a standard nick translation protocol and hybridized to chromosomes isolated from peripheral blood lymphocytes. Probe detection was performed using tetramethylrhodamine
isothiocyanate-labeled antibodies. To further define the regional
localization, chromosome banding was performed (21).
Isoform Analysis by PCR--
A series of primers distinguishing
the apoEr2 isoforms by size were utilized to analyze cell type-specific
expression. Primer positions are indicated in Fig. 4. Each experiment
was carried out with outer primers for the first amplification and
inner primers for nested PCR. Outer primer pairs were primer A
(5'-AAGGACTGCGAGGGTGGAGCG-3', nucleotides 454-474) and primer B (5'
-CCAGCAACCAAACATCTTCTG-3', nucleotides 3093-3114). Primer L5
(5'-ACATGTGTCCTTGCAATCAAGCAC-3', nucleotides 541-565) and primer L3
(5'-GGATGGACAAGCTCTTAGCAC-3', nucleotides 938-959), primer O5
(5'-ATCTGGGCTCAACGGTGTGGAC-3', nucleotides 1653-1671) and primer O3
(5'-GGCTATCACCACTATGGGCACG-3', nucleotides 2441-2463), primer C5 and
primer C3 5'-GATCCCATCCTCAGGGTAGTCC-3', nucleotides 2829-2851) were
utilized as inner primers. cDNAs were reverse-transcribed from
cultured HUVEC, HSMEC, SMC, and systemic artery mRNAs,
respectively. Oligo(dT) primers served as templates for PCR
amplification (40 s at 94 °C, 40 s at 58 °C, and 1 min at
68 °C, 25 cycles). Negative controls in the absence of reverse transcriptase were included in the analysis. All products were sequenced.
To confirm alternative splicing of the novel sequences at the level of
genomic DNA, we used primer pair L5 and 155 (5'-GAGGGTCAGAGCAGAAAGTGTC-3', nucleotides 833-854 of
apoEr2906) employing 250 ng of human chromosomal DNA
purified from peripheral blood lymphocytes (QIAamp blood kit, Qiagen)
as PCR template.
In Situ Hybridization--
Nonradioactive in situ
hybridization was performed on cryosections (7 µm) of neuronal
tissues obtained from a rhesus monkey tissue bank (22). Three
riboprobes covering specific regions of the new splice variants of the
human apoEr2 mRNA found by us (Fig. 4) were transcribed from the
respective vectors and designated as 1) "ligand binding repeat
region" (nucleotides 616-956; 340 base pairs long), 2)
"O-linked sugar domain region" (nucleotides 2085-2310;
225 base pairs long long), and 3) "cytoplasmatic tail region"
(nucleotides 2629-2809; 180 base pairs long) using the digoxigenin-RNA
labeling kit (Roche Molecular Biochemicals).
In brief, slides were fixed in buffered, freshly prepared 4%
paraformaldehyde solution for 10 min and then incubated in PBS containing 100 mM glycine and PBS containing 0.3% Triton
X-100. After a short washing step with PBS, sections were digested for 30 min at 37 °C with TE buffer (100 mM Tris-HCL, 50 mM EDTA, pH 8.0) containing 1 mg/ml RNase-free proteinase
K. Sections were again postfixed in 4% paraformaldehyde, washed in
PBS, and treated in triethanolamine/acetic anhydride solution (0.1 M triethanolamine, pH 8.0, containing 0.25% acetic
anhydride) on a rocking platform. After another PBS washing step, the
slides were wiped dry around the tissue and laid out flat in airtight
boxes on top of filter paper soaked in box buffer (4× SSC, 50%
formamide). Each section was covered with 20 µl of prehybridization
buffer (50% formamide, 0.6 M NaCl, 10 mM
Tris-HCL, pH 7.5, 1 mM EDTA, 50 mg/ml heparin, 10 mM dithiothreitol, 10% polyethylene glycol 8000, and
Denhardt's solution). 7 µl of riboprobe in 40 µl of hybridization
buffer were added to each section and incubated for 16 h at
52 °C. Hybridization was followed by two washes (10 min each) at
room temperature in wash buffer (2× SSC, 10 mM
2-mercaptoethanol, 1 mM EDTA). Slides were then immersed in
RNase A solution (20 mg/ml) for 30 min at 37 °C and washed again
twice in wash buffer at room temperature then for 2 h in wash
buffer containing 0.1× SSC at 55 °C. Detection of the labeled and
hybridized probe was performed using the DIG nucleic acid
detection kit (Roche Molecular Biochemicals) by incubation for 30 min
with alkaline phosphatase-labeled anti-digoxigenin Fab fragment (Roche
Molecular Biochemicals, 1:700 in 0.1% goat serum, Tris-buffered saline
at room temperature); the signal was developed using nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate.
Consecutive sections were analyzed the three different antisense probes
as well as the respective sense probe as control for nonspecific hybridization.
Identification of New Splice Variants of apoER2
mRNA--
The degenerate upstream primer LDLRF31,
corresponding to the coated pit signal Asn-Pro-Val-Tyr (NPVY) within
the cytoplasmic tail (23), and the degenerate downstream primer
LDLRF50, corresponding to the tetrapeptide sequence Ala-Val-Asp-Trp
(AVDW) within the EGF precursor domain, were used for initial reverse
transcriptase-PCR experiments. Inosine was utilized in each position
having more than two bases degeneracy. Using this strategy, a LDLR
fragment of 1070 bp and a VLDLR fragment of 980 bp were readily
identified in cultured HUVEC, HSMEC, and SMC. In all three cell types,
two new PCR fragments (1115 and 889 bp) were obtained (Product
1 and Product 2 in Fig.
1A). The fragment
corresponding to LRP (4200 bp) could only be detected in SMC utilizing
a semi-nested PCR strategy (data not shown). All four PCR products
shown in Fig. 1A were sequenced and confirmed as LDLR and
VLDLR, respectively, containing the 84-bp O-linked sugar
domain (VLDLR-2). The two novel fragments with partial sequence
identity carried an open reading frame corresponding to an EGF
precursor region with YWTD motifs and a transmembrane domain. The two
clones differed by 225 bp upstream of the transmembrane domain that
were similar to the O-linked sugar domain of LDLR and VLDLR.
Northern blot analysis (Fig. 1B) utilizing the 1115-bp
fragment as a probe yielded multiple transcripts corresponding to the
4.5, 3.9, and 3.6-kb mRNA species, respectively, in HUVEC, HSMEC,
and SMC. In comparison, the VLDLR probe hybridized to a 4.0- and a
5.5-kb transcript and the LDLR probe to a 5.3-kb transcript, which were
all distinct from the transcripts detected with the 1115-bp probe.
PCR-Cloning and Sequence Analysis--
Amplification of the 5'-end
was achieved by combining the degenerate downstream primer LDLRF51
corresponding to the SDE sequence within the ligand binding region and
a specific upstream primer (primer 5) overlapping the novel sequence by
80 bp. Using the 5'- and 3'-rapid amplification of cDNA ends
technique with specific primers 90 bp downstream and 150 bp upstream of
the sequence, the missing 5'- and 3'-ends of the cDNA were
identified. One large open reading frame was identified coding for a
protein with 906 amino acids and corresponding to a calculated
molecular mass of 100 kDa (Fig. 2). The
sequence was partially identical to sequences of the originally
described apoEr2. The new sequence found was different from
apoEr2922 by lacking repeats 4-6 of the ligand binding
domain and containing additional sequences homologous to ligand
binding repeat 8 of VLDLR and an additional cysteine-rich region with
no homology to any sequence of a member of the low density lipoprotein
receptor family.
Chromosomal Localization and Genomic Organization--
To clarify
the nature of the various forms with partial identity to apoEr2,
genomic analysis was performed using nucleotides 1444-2558 of apoEr2
common to all forms (Fig. 3). This
sequence could be localized as a single copy on human chromosome 1p32
by fluorescence in situ hybridization (Fig. 3). Kim et
al. (24) identify the apoEr2 gene on chromosome 1p34, suggesting
that the different forms found by us are splice variants of apoEr2.
Exon 6 of apoEr2 was found to be separated from the novel exon 6a by a
~0.75-kb intron. Both the ligand binding repeat 8 and the novel cysteine-rich region of apoEr2906 are encoded by this exon
located within the 3.7-kb intron region identified by Kim et
al. (24). The exon-intron junctions conform to the GT/AG rule (25)
(Fig. 2).
Structural Comparison of the Splice Variants of
ApoEr2--
Protein domains in alignment with the exon organization
are shown in Fig. 4. In theory, 12 apoEr2
isoforms could result from the combinations of the various alternative
regions. The shortest apoEr2 mRNA isoform apoEr2659
encodes four ligand binding repeats, an EGF precursor domain, a
transmembrane domain, and a short cytoplasmic region (isoform not shown
in Fig. 4). The apoEr2906 variant consists of five ligand
binding repeats, the novel cysteine-rich domain, an EGF precursor
domain, an O-linked sugar domain, a transmembrane domain,
and a cytoplasmic region. Additional alternative forms of apoEr2, for
example isoforms containing the O-linked sugar domain and
the 59-amino acid deletion within the cytoplasmic region, are shown in
Fig. 4. The isoforms apoEr2718 and apoEr2
apoEr2734, both lacking the ligand binding repeat 4-6,
were also described recently by Clatworthy et al.
(16).
Tissue-specific Expression of Splice Variants of
ApoER2906 mRNA and Expression in Cultured
Cells--
To analyze which splice variants are actually expressed in
different tissues, a reverse transcriptase-PCR strategy with several primer combinations as presented in Fig.
5A was used. Splicing-mediated insertion or deletion of sequence(s) is revealed by generation of
multiple PCR fragments of different sizes (Fig. 5B). As
expected, mRNA of the novel ligand binding repeat and the novel
cysteine-rich domain were present in HUVEC, HSMEC, and also in SMC and
placenta, whereas none of the other tissues contained these alternative regions. HUVEC and SMC were found to express an apoEr2 mRNA isoform containing the novel cysteine-rich region but not the novel ligand binding repeat (Fig. 5B, 1, apoEr2710
as shown in Fig. 4). In brain and placenta, the predominant products
contained the O-linked sugar domain, whereas in heart, an
exclusive PCR product lacking the O-linked sugar domain was
detected (Fig. 5B, 2). Both alternate cytoplasmic forms were
identified in placenta. In heart, the predominant mRNA product
contained the 59-amino acid insertion, whereas in brain, the shorter
product was detected (Fig. 5B, 3). ApoEr2
mRNA containing the cytoplasmic region without the 59-amino acid
insertion and the isoform with and the isoform lacking the
O-linked sugar domain was identified in coronary arteries
from healthy individuals (data not shown).
In Situ Hybridization of Different Splice Variants of ApoEr2
mRNA--
To support the tissue PCR data by an independent method,
we used in situ hybridization specific for different splice
variants of apoEr2 mRNA. We chose brain tissue because of the high
apoEr2 expression in human brain observed by our own Northern analysis and because of the existing in situ hybridization data for
the rat brain (15), human brain (16), and mouse brain (25). Cerebral tissue sections of rhesus monkey were employed together with
digoxigenin-labeled probes specific for the different splice variants
of apoEr2 mRNA. We could detect the same differences in the
expression pattern between the different splice forms of the apoEr2
mRNAs as revealed from PCR experiments. Fig.
6 shows the expression pattern for the
riboprobes specific for the newly identified ligand binding domain, the
O-linked sugar domain, and the cytoplasmic tail in the
central nervous system (Fig. 6, A-C). Using the riboprobe
specific for the new ligand-binding site (Fig. 6A), we could
not detect any signal in neuronal tissue but only strong expression in
vascular cells (inset). We observed a similar mRNA
expression pattern using the other two riboprobes specific for the
O-linked sugar domain (Fig. 6B) and the
cytoplasmic tail (Fig. 6C), for which large and small
neurons were found to be positive. In vessels, the endothelium
expressed all three splice variants analyzed in a similar pattern
(insets in Fig. 6, A-C). The negative control
using a sense strand probe was always found negative (not shown). In
other parts of the brain (not shown), the O-linked sugar
domain and the cytoplasmic tail but not the new ligand binding domain
were found positive in several types of cells. In the cerebellum
Purkinje cells, the large neurons located at the border of the granular
and the molecular layer expressed high levels, whereas the basket cells
and the granule cells show low levels of these two apoER2 mRNA
variants. Furthermore the oligodendrocytes in the white matter
expressed low levels of these two splice variants of the apoEr2
mRNA.
In search for novel members of the LDLR family in vascular tissue,
we found several hitherto unknown mRNA transcripts using a
homology-cloning strategy. Analyzing the origin of these novel transcripts, they were found to be splice variants of the apoEr2 (15).
Theoretically, 12 apoEr2 isoforms could result from the combinations of
the various alternative regions. ApoEr2906 lacks ligand
binding repeats 4-6 (nucleotides 497-883 of apoE2) and, therefore,
the short linker region between ligand binding repeats 5 and 6, thought
to be required for low density lipoprotein binding by LDLR (8) is
absent. The fifth ligand binding repeat of apoEr2906 exhibits the highest homology (55%) to ligand binding repeat 8 of
VLDLR. A comparable ligand binding repeat has recently been described
within the chicken lipoprotein receptor LR8B (27), which has high
sequence homology to apoEr2. The cysteine-rich domain of
apoEr2906 is a novel sequence with no homology to any known
protein. It is important to note that these two regions each contain an
odd number of cysteines (n = 5), suggesting tertiary structural disulfide bridge formation. Another AG nucleotide sequence within exon 6a gives rise to a possible additional alternative spliced
variant with a truncated cysteine-rich region encoding for four
cysteines (not shown). Whether new motifs with new ligand binding
properties result from formation of disulfide bonds between cysteines
of adjacent domains in addition to the possible variation in ligand
binding properties caused by alternative splicing remains to be
determined. Data from fluorescence in situ
hybridization localizing apoEr2 on chromosome 1p32 indicate that the
novel alternative transcripts and the original apoEr2 mRNA are
alternatively spliced variants of the same gene locus, since Kim
et al (24) identify the apoEr2 gene on chromosome 1p34.
Previously published data have already indicated
alternative splicing of the O-linked sugar domain of VLDLR
(28) and chicken lipoprotein receptor 8 (LR8) (27) as well as of the
apoEr2 itself (16). The intracellular domain of apoEr2 contains motifs
that have previously been described in human gp330 (29). The
alternatively spliced cytoplasmic region of human apoEr2 contains the
amino acid sequence motifs LPGEPRS and LPKNPLS, with two potential Src homology 3 binding regions (30, 31), indicating a possible intracellular signaling through these sites. In contrast, these motifs
are not present in LDLR, VLDLR, or LRP.
Our data revealed by in situ hybridization confirm and
expand with single cell resolution earlier results by Kim et
al. (15) obtained with radioactive in situ
hybridization. The data are also consistent with results on normal
mouse embryo brain, published by Trommsdorff et al. (26) and
with the detection of apoEr2 in human brain (16). These authors also
could not detect splice variants containing the ligand binding domain
4-6 of apoEr2 within the brain, consistent with our data. To assure
that comparable parts of tissue were analyzed, consecutive sections
were used for the three different riboprobes and the respective sense controls.
In conclusion, we have identified a family of new isoforms of apoEr2
mRNAs originating from alternative splicing. The restriction of the
expression of a mRNA isoform coding for a new ligand binding domain
in brain tissue to vascular cells could indicate possible binding of a
specific ligand only to vascular cells but not to neuronal tissue. This
could indicate a specific function of this splice variant in vascular
cells or, alternatively, disruption of the vascular cell-specific
expression and expression also in neuronal tissue and, in turn, uptake
of a novel ligand into neuronal cells normally not internalized could
contribute to brain pathologies (15).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection of apoEr2 mRNA in EC and
SMC. Panel A, amplification with LDLR family specific
primers as described under "Experimental Procedures." PCR
products corresponding to LDLR and VLDLR and two additional bands were
observed: Product 1, a 1115-bp band; Product 2, a 889-bp band with high
sequence homology to VLDLR and LDLR. VLDLR-2 represents the
VLDLR-specific PCR product containing the O-linked sugar
domain. Panel B, identification of multiple variants of
apoEr2 mRNA by Northern blotting. The blotted mRNAs were first
hybridized with a probe corresponding to labeled Product 1 and then
rehybridized with a VLDLR, a LDLR, and a rat glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) probe. Arrows indicate
transcript sizes.
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Fig. 2.
Nucleotide sequences and deduced amino acid
sequence of human apoEr2906. The signal sequence and
the transmembrane sequence are boxed. The internalization
motif is given in bold underline. The O-linked
sugar domain (between nucleotides 2162 and 2386) is given in
bold. Cysteine residues are circled.
Arrowheads indicating the start and end of alternatively
spliced regions mark alternative splicing sites. The first
arrowhead marks the site of ligand binding repeats 4-6 of the
published apoEr2 sequence (15). The nucleotide stretch between
arrowheads 2-5 represents transcripts of novel exon 6a as
indicated in gray in Fig. 3. Arrowhead 3 points
to another alternative splicing site that has been identified but is
not further discussed. Arrowhead 4 corresponds to the splice
site generating the apoEr2710 isoform shown in Fig.
3. Underlining highlights the four binding sites of
the degenerate primers.
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Fig. 3.
Fluorescent in situ
hybridization of labeled human apoEr2 cDNA (nucleotides
1444-2558) to R-banded human metaphase chromosomes. A schematic
representation of human chromosome 1 is shown on the
right.
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Fig. 4.
Protein domains of ApoEr2 isoforms in
alignment with the exon organization. The ligand binding repeats
are numbered, and the growth factor repeats are indicated by
capital letters. Arrowheads mark the position of
the introns. The bold line separating ligand binding repeats
five and six in apoEr2922 indicates the linker region.
Transmembrane stands for transmembrane domain,
cytoplasmic represents the cytoplasmic region,
Cysteine-rich indicates the cysteine-rich region,
ligand binding indicates the ligand binding regions, and
EGF precursor homology stands for the EGF precursor
homology-region.
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Fig. 5.
Structural characterization of
apoEr2906 mRNA isoforms. Panel A, schematic
drawing of apoEr2906 mRNA, illustrating alternatively
expressed regions, with the positions of the specific primers used for
PCR analysis. Panel B, 1, PCR products obtained
with primers L5 and L3, resulting in the amplification of ligand
binding repeats 4 and 5 and the novel cysteine-rich region of
apoEr2906. Panel B, 2, PCR products
obtained with primers O5 and O3 flanking the alternatively spliced
O-linked sugar domain. Panel B, 3, PCR products
obtained from amplification with primers flanking the alternatively
spliced cytoplasmic region.
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Fig. 6.
In situ hybridization in brain
tissue (brain stem). A-C, nonradioactive in
situ hybridization of the different apoEr2 mRNAs using
digoxigenin-labeled RNA antisense strand probes visualized by
phosphatase-labeled anti-digoxigenin antibodies. A, the
riboprobe, specific for the ligand binding domain, did not reveal any
positive staining in neuronal cells but showed positive signals within
the endothelium of a small vessel (inset). B and
C, hybridization of the riboprobes specific for the
O-linked sugar domain (B) and the cytoplasmic
tail (C) in several small and large neurons; in endothelium,
the same positive signal for all three different riboprobes is seen
(inset in B and C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We appreciate the expert technical help of Thomas Nardelli in the preparation of the artwork.
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FOOTNOTES |
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* This work was supported in part by Austrian Science Foundation Grants F509 and P10559. An abstract of this article has been published (Korschineck, I., Ziegler, S., Breuss, J., Lang, I., Lorenz, M., Kaun, C., Ambros, P. F., and Binder, B. R. (1997) Thromb. Haemostasis 77, Suppl., p. 400, Abstr. OC1630.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) Z75190.
§ These authors contributed equally to this report.
** To whom correspondence should be addressed. Tel.: 43-1-427762501; Fax: 43-1-42779625; E-mail: Bernd.Binder@univie.ac.at.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M011795200
2 Nucleotide numbering corresponds to the sequence shown in Fig. 2.
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ABBREVIATIONS |
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The abbreviations used are: LDLR, low density lipoprotein receptor; apoEr2, apolipoprotein E receptor 2; LRP, LDLR-related protein; VLDLR, very low density lipoprotein receptor; SMC, smooth muscle cells; EGF, epidermal growth factor; EC, endothelial cells; HUVEC, human umbilical vein EC; HSMEC, human skin microvascular EC; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s).
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REFERENCES |
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