(Received for publication, December 20, 1996)
From the Apolipoprotein E receptor 2 is a recently
identified receptor that resembles low and very low density lipoprotein
receptors. Isolation and characterization of genomic clones encoding
human apolipoprotein E receptor 2 revealed that the gene spans ~60
kilobases and contains 19 exons. The positions of the exon/intron
boundaries of the gene are almost identical to those of low and very
low density lipoprotein receptors. Fluorescent in situ
hybridization of human chromosomes revealed that the gene is located on
chromosome 1p34. Isolation of a cDNA encoding a variant receptor
and reverse transcription-polymerase chain reaction indicate the
presence of multiple variants with different numbers of cysteine-rich
repeats in the binding domain of the receptor. We also found a variant receptor lacking a 59-amino acid insertion in the cytoplasmic domain.
The transcription start site was mapped to the position 236 base pairs
upstream of the AUG translation initiator codon by primer extension
analysis. Sequence inspection of the 5 Apolipoprotein E (apoE)1 is a 34-kDa
lipophilic protein that circulates in the plasma primarily as a major
component of various lipoproteins including chylomicron remnants,
intermediate density lipoprotein, very low density lipoprotein (VLDL),
ApoE also has functions in the central nervous system (reviewed in
Refs. 3 and 4). Although the major site of apoE synthesis is the liver,
the brain contains the second highest abundance of apoE mRNA (5).
ApoE synthesis is dramatically increased after injury of the rat
sciatic or optic nerves (6). In the brain, significant concentrations
of apoE are detected in astrocytes, including Bergmann's glia of the
cerebellum, tanycytes of the third ventricle, pituicytes of the
neurohypophysis, and Müller cells of the retina (7). These
results indicate that apoE may be involved in the mobilization and
utilization of lipid in the central nervous system. In humans, there
are three major isoforms of apoE, designated E2 (Cys112 and
Cys158), E3 (Cys112 and Arg158),
and E4 (Arg112 and Arg158), which are products
of three alleles at a single gene locus. Genetic data indicate that the
e4 allele is present with increased frequency in patients with sporadic
(8) and late-onset familial (9) Alzheimer's disease.
In previous studies, we have isolated a human cDNA encoding a novel
receptor that binds apoE-rich ApoER2 mRNA is detectable most intensely in brain and testis and,
to a much lesser extent, in ovary, but is undetectable in other tissues
in rabbit (10). In human tissues, apoER2 mRNA is abundant in brain
and placenta and undetectable in other tissues. This pattern of tissue
distribution and the relative abundance of apoER2 mRNA are
completely different from those of LDLR and VLDLR: VLDLR mRNA is
most highly expressed in heart and muscle (12), whereas LDLR mRNA
is expressed in various tissues including liver (14). This pattern of
tissue expression of apoER2 mRNA suggests that the receptor plays a
role in the uptake of apoE containing high density lipoprotein secreted
from astrocytes in the central nervous system.
Recently, Novak et al. (15) have identified a novel LDLR
homologue with an 8-fold cysteine-rich repeat predominantly expressed in chicken brain. This chicken protein, designated LR8B, consists of
five domains resembling those of LDLR, VLDLR, and apoER2. Comparison of
the amino acid sequence of LR8B with those of human LDLR, VLDLR, and
apoER2 reveals that it is a chicken homologue of apoER2: the two
proteins have ~77% of their amino acids in common, and the identities extend throughout the proteins, excluding an extra cysteine-rich repeat present in LR8B and an insertion sequence present
in human apoER2. The presence of apoER2 in chicken is striking because
birds are not known to synthesize apoE (16, 17).
To clarify the structural and functional relationships of apoER2 and as
an initial approach to study the mechanisms regulating apoER2 gene
expression, we have cloned and characterized the human gene encoding
apoER2. In this paper, we describe the exon/intron organization,
chromosome location, and transcription units of the human apoER2 gene.
We also present evidence for the presence of multiple forms of variant
receptor generated by alternative splicing.
Unless otherwise indicated, all restriction and
DNA-modifying enzymes were from Takara Shuzo Corp. (Kyoto, Japan).
[ Standard molecular biology techniques were
performed essentially as described by Sambrook et al. (18).
cDNA and genomic clones were subcloned into pBluescript vectors in
both orientations and sequenced by the dideoxy chain termination method
(19) manually or on an Applied Biosystems Model 373A DNA sequencer.
Large DNA fragments were shortened successively by exonuclease III (20) and subcloned into pBluescript vectors.
Recombinant bacteriophage
clones were isolated by plaque hybridization from a library of human
normal peripheral leukocytes in Human/rodent somatic cell
hybrid mapping panel 2 of the National Institute of General Medical
Sciences (Coriell Institute for Medical Research, Camden, NJ) was
analyzed by PCR to assign the chromosome location of the human apoER2
gene. PCR oligonucleotide primers were synthesized as follows: forward
primer, 5 The human apoER2 cDNA
was digested with SalI and labeled with biotin-16-dUTP
(Boehringer Mannheim) by nick translation. Fluorescent in
situ hybridization to bromodeoxyuridine-synchronized metaphase chromosomes was carried out as described previously (23). For fluorochrome detection, slides were incubated with fluorescein isothiocyanate-conjugated avidin DCS (Vector Laboratories, Inc.). The
fluorescein isothiocyanate signals were amplified by incubation with
biotin-conjugated goat anti-avidin D antibody (Vector Laboratories, Inc.), followed by a second incubation with fluorescein
isothiocyanate-conjugated avidin DCS. The preparations were
counterstained with propidium iodine and examined in a Zeiss laser
scanning microscope. At least 75 metaphases were analyzed.
Rabbit cDNA was synthesized from 1 µg of poly(A) RNA
from human brain (CLONTECH) or placenta using 200 units of Superscript
(Life Technologies, Inc.) and random hexamers in 20 µl of standard
reverse transcription buffer (50 mM Tris-HCl, pH 8.3, 10 mM MgCl2, 50 mM KCl, 3 mM dithiothreitol, 0.1% Nonidet P-40, and 0.45 mM dNTP) at 45 °C for 1 h. 1-µl aliquots of the
reaction mixture were then subjected to "hot start" PCR using
AmpliTaq Gold (Perkin-Elmer) and a set of primers corresponding to the
ligand-binding domain (sense primer: oligonucleotide 24, 5 An oligonucleotide
(oligonucleotide 180R, 5 To test for promoter
activity, various lengths of the 5 HepG2 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. PC12
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 5% fetal bovine serum, 5% horse serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin. Cells were transfected with
1.5 µg of test plasmid and 0.5 µg of the Transfected cells were washed three times
with phosphate-buffered saline; lysed in 500 µl of 25 mM
Tris phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM CDTA, 10% glycerol, and 0.1% Triton X-100; and
centrifuged to remove cell debris. An aliquot of 20 µl of the cell
extract was incubated in the presence of ATP, luciferin, and coenzyme A
(28), and light emission was measured using a Berthold Lumat LB 9501 luminometer. The protein content of the cell extract was measured by
the method of Lowry et al. (29).
An aliquot of the cell extract from
lysed transfected cells was incubated at 37 °C for 1 h with
4.85 mg/ml chlorophenol red- The restriction map of a
60-kb region containing the human apoER2 gene was constructed by
analysis of the four bacteria phage clones
Exon/intron organization of the human apoER2 gene
Exon sequences are in upper-case letters; intron sequences are in
lower-case letters. Introns are positioned by applying the GT/AG rule
(31).
Tohoku University Gene Research Center,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-flanking region revealed
potential DNA elements: AP-2, GC factor, PEA3, and Sp1. The minimal
promoter region and a region required for nerve growth factor
inducibility in PC12 cells were also determined.
-migrating VLDL (
-VLDL), and high density lipoprotein (with apoE)
(reviewed in Ref. 1). It is a key molecule responsible for the cellular recognition and internalization of these lipoproteins. Biochemical and
genetic studies have demonstrated that apoE is involved in the hepatic
clearance of chylomicron remnants and VLDL remnants from the plasma
(reviewed in Refs. 1 and 2).
-VLDL with high affinity and
internalizes it into the cells (10). This new receptor, designated apoE
receptor 2 (apoER2), consists of five domains that resemble those of
the low density lipoprotein receptor (LDLR) (11) and the VLDL receptor
(VLDLR) (12, 13): (i) an amino-terminal ligand-binding domain composed
of multiple cysteine-rich repeats, (ii) an epidermal growth factor
precursor homology domain, (iii) an O-linked sugar domain
with clustered serine and threonine, (iv) a transmembrane domain, and
(v) a cytoplasmic domain with an FDNPVY sequence (14). The structural
features of each domain of apoER2 are highly similar to those of LDLR
(11) and VLDLR (12, 13). A key structural difference among the three
receptors is the number of cysteine-rich repeat sequences in their
ligand-binding domains: apoER2 and LDLR contain a 7-fold repeat,
whereas that of VLDLR is 8-fold. Although apoER2 and LDLR contain the
same number of cysteine-rich repeats, the ligand-binding domain
structure of apoER2 is much more closely related to that of VLDLR:
apoER2 and VLDLR contain a short linker sequence between repeats 5 and 6, whereas that of LDLR is located between repeats 4 and 5.
Materials
-32P]ATP (3000 Ci/mmol) and
[
-32P]dCTP (3000 Ci/mmol) were from Amersham Corp.
Oligonucleotides were synthesized with an automated DNA synthesizer
(Applied Biosystems Inc., Model 381A).
EMBL3 vector (13) using the entire
coding region of the human apoER2 cDNA (10) labeled with
[
-32P]dCTP by random priming methods (21). Through
screening of 2 × 106 clones, we obtained 24 positive
clones, of which four (
NR7,
NR10,
NR19, and
NR22) were
chosen for further analysis. Intron sizes were determined by Southern
blotting, restriction mapping, and polymerase chain reaction (PCR)
analysis (22) of the genomic clones. DNA fragments carrying exons were
identified by restriction mapping and Southern blotting. After
subcloning into pBluescript vectors, the sequences of exons,
exon/intron boundaries, and the 5
-flanking region were determined.
Gaps between
NR7 and
NR10 and between
NR10 and
NR22 could
not be isolated after several attempts to screen the genomic library
with region-specific probes. The sizes of the gaps between
NR7 and
NR10 and between
NR10 and
NR22 were determined by Southern
blotting and PCR with exon-specific oligonucleotides.
-AGT CCC ATG CAC TAC ACT CTG G-3
; and reverse primer,
5
-TGA GGG TGA GGC ATA TCC TAT C-3
. Both primers are contained
within exon 19 and are specific to the human apoER2 sequence. The PCR
amplification reactions consisted of 33 cycles of 94 °C for 30 s, annealing at 57 °C for 2 min, and extension at 72 °C for 3 min. PCRs were performed using 100 ng of control human, mouse, and
hamster genomic DNAs to confirm that the primers were specific for
human DNA. Genomic DNA (100 ng) from each panel cell line was
amplified, and the chromosomal assignment was performed by use of
concordance tables.
-VLDL (d
1.006 g/ml) was prepared from the plasma of 1% cholesterol-fed animals
(24). 125I-Labeled
-VLDL was prepared as described (25).
Binding of 125I-labeled
-VLDL by the transfected
LDLR-deficient ldlA-7 cells (26) was assayed according to
the procedure described by Kim et al. (10).
-TCT CCG GCT TCT GGC GCT-3
(25 pmol); and antisense primer:
oligonucleotide 1114, 5
-TCT GGT CCA GGA GCT GGA A-3
(25 pmol))
in a total volume of 100 µl. After heating at 94 °C for 10 min,
amplification proceeded for 33 cycles, with denaturation for 30 s
at 94 °C, annealing of primers for 1 min at 63 °C, and extension
for 90 s at 72 °C. This was followed by a final extension step
at 72 °C for 10 min. To amplify the region corresponding to the
cytoplasmic domain, PCR was carried out under standard conditions for
33 cycles with primer annealing at 63 °C in a total volume of 100 µl (sense primer: oligonucleotide 2546, 5
-GAA ACT GGA AGC GGA AGA AC-3
(25 pmol); and antisense primer:
oligonucleotide 2918, 5
-GAG GCA CGA AGG GGG TGA T-3
(25 pmol)). The PCR products were analyzed by electrophoresis on a 2%
agarose gel.
-TCT CAG CCC TCC GAG TCC TTG-3
)
complementary to the 5
-end of the human apoER2 mRNA was
end-labeled with [
-32P]ATP. 5 × 105
cpm of primer was coprecipitated with either 1 µg of poly(A) RNA or
15 µg of yeast tRNA. RNA and primer were resuspended in 100 µl of
standard reverse transcription buffer and heated at 95 °C for 1 min.
cDNA synthesis was carried out using 200 units of Superscript at
45 °C for 1 h. Primer extension products were analyzed on 6%
denaturing polyacrylamide gels adjacent to dideoxynucleotide chain
termination sequencing ladders derived from double-stranded genomic DNA
fragments using the same primer. To confirm the results of primer
extension analysis, RT-PCR (see above) was carried out with
combinations of an antisense primer (oligonucleotide R,
5
-GCC GCC GAG CAG CGG ATC AGC-3
(25 pmol)) and three sense
primers (oligonucleotide A, 5
-TGA GAG AAG AGT GGA CGA AAG AC-3
(25 pmol); oligonucleotide B,
5
-AAC CTG CTT GAA ATG CAG CCG AG-3
(25 pmol); and
oligonucleotide C, 5
-GCA AGG ACT CGG AGG GCT-3
(25 pmol)). PCR
was carried out under standard conditions. The thermal profile used was
94 °C for 30 s, 60 °C for 1 min, and then 72 °C for
2 min.
-upstream regions of exon 1 were
fused to the luciferase gene present in pPGV-B (ToyoInki Inc., Tokyo).
This plasmid contains no eukaryotic promoter or enhancer elements. DNA
fragments containing nucleotides
437 to +8 or nucleotides
316 to +8
were generated by exonuclease III, blunt-ended with Klenow fragment,
followed by digestion with NotI, and then inserted into the
SmaI/NotI sites of pPGV-B to create reporter
plasmids pLAER437 and pLAER316, respectively. A reporter plasmid
containing nucleotides
148 to +8 (pLAER148) was created by insertion
of the 156-bp PstI/NotI fragment of pLAER437 into
the PstI/NotI sites of pPGV-B. The sequences of
the inserts of these reporter plasmids were confirmed by nucleotide
sequencing.
-galactosidase
expression plasmid pCMV
(27) using LipofectAMINE (Life Technologies
Inc.) reagent according to the manufacturer's recommendations. Cells
were harvested 48 h after transfections for the measurement of
luciferase activities. PC12 cells were cultured in the presence or
absence of 50 ng/ml nerve growth factor (NGF) for 3 h before
harvest for luciferase assay. In each transfection experiment, parallel
plates of HepG2 and PC12 cells were transfected with pPGV-B and pPGV-C
(ToyoInki Inc.), which serve as negative and positive controls,
respectively. pPGV-B lacks a eukaryotic promoter, and apparently no
luciferase activity was detected in both cell lines transfected with
pPGV-B. The pPGV-C plasmid contains the SV40 early promoter and
enhancer driving the expression of the luciferase mRNA
transcript.
-Galactosidase Assay
-D-galactopyranoside (Boehringer Mannheim), 62.3 mM MgCl2, and 45 mM
-mercaptoethanol. The reaction was stopped with 0.5 ml of 1 M Na2CO3, and the amount of
chlorophenol red formed was measured spectrophotometrically at 575 nm
(30). To normalize the transfection efficiency for each individual
transfection, the luciferase activity expressed as integrated light
output values/mg of protein was divided by the
-galactosidase
activity expressed as units/mg of protein.
Isolation of the Human ApoER2 Gene
NR7,
NR10,
NR19,
and
NR22 (Fig. 1). Gaps between
NR7 and
NR10
and between
NR10 and
NR22 were estimated to be ~3 kb and 1 kb,
respectively, based on the analysis of genomic DNA by Southern blotting
and PCR with exon-specific oligonucleotides. The exact exon/intron
boundaries were determined by nucleotide sequencing. Analysis of these
genomic clones revealed that the gene spans ~60 kb and contains 19 exons (Fig. 1). Sequences at all the exon/intron junctions (Table
I) conformed to the GT/AG rule (31).
Fig. 1.
Schematic representation of the apoER2 gene.
A, restriction map of the human apoER2 gene. The cleavage
sites for EcoRI and XhoI are shown. B,
the four genomic clones used for analysis of the apoER2 gene. NR19
and
NR22 are overlapping clones. There is no overlapping between
NR7 and
NR10 or between
NR10 and
NR22. The gaps between
NR7 and
NR10 and between
NR10 and
NR22 are ~3 kb and 1 kb, respectively, as determined by Southern blotting and PCR analysis
of genomic DNA. C, the sequencing strategy. Exons are
represented by closed boxes and are numbered.
Arrows indicate the extent and direction of sequence
reactions. D, diagram of the apoER2 gene. Exons are denoted
by closed boxes and are numbered. Introns are
indicated by open boxes.
[View Larger Version of this Image (20K GIF file)]
Exon No.
Exon size
Sequence at exon/intron
junction
Amino acid interrupted
Intron length
5
-Splice
donor
3
-Splice acceptor
bp
kb
G Q G
P A
1
360
GGCCAAG gtgcgtgcagcc
tccccctcgcag GGCCGGCC
Gly1
0.8
D C P
K K
2
120
GACTGCC gtgagtggcggg
cttgccctgcag CCAAGAAG
Pro41
13.0
T C T
T K
3
123
ACTTGCA gtgagtcctgcc
ctgtctccacag CCAAGCAG
Thr82
9.0
A T L
C A
4
129
GCTACCT gtgagtctgggg
tgtcccgcgcag TGTGCGCC
Leu125
3.2
D C P
L G
5
387
GACTGCC gtaagccccttc
ctctcctggcag CACTGGGC
Pro254
1.0
L Q G
L N
6
123
CTACAGG gtgcgtgtggtc
gtccctccccag GGCTGAAC
Gly295
3.7
C G D
I D
7
120
TGTGGCG gtgagacctcct
tcctccccacag ACATTGAT
Asp335
0.1
A A G
G K
8
126
GCTGCTG gtatgaacaccc
cctgcatgacag GTGGAAAG
Gly387
3.6
I Y S
A Y
9
175
ATCTATAG gtgagcggccat
ttttctcttaag CGCCTAC
Ser435
2.0
L R G
F M
10
228
CTGCGAGG gtgagtatccca
tttgtcttttag GTTCATG
Gly511
1.6
T L D
L L
11
119
ACCCTGG gtgagccctgcc
ttccttcccaag ATCTGCTG
Asp551
0.3
V F E
D K V
12
140
GTGTTTGAG gtgagtcctgta
tataccccacag GACAAGGTG
Glu587
1.6
P R A
P D
13
142
CCAAGAG gtgagctgtctc
ctctgccattag CTCCAGAT
Ala645
2.1
Y R A
P Q
14
153
TACCGAG gtaagcagacct
tttcccctccag CACCTCAA
Ala696
0.8
Q H Y
A N
15
225
CAGCACT gtaaggaaatga
tgtgttttacag ATGCAAAT
Tyr771
1.7
P I V
V I
16
69
CCCATAG gtgagtgtggct
tccttcctccag TGGTGATA
Val794
3.8
Y P A
A I S
17
173
TATCCTGCA gtaagtatttct
tgggggtgtcag GCAATCAGC
Ala851
0.9
K S K
R V A
18
177
AAATCCAAG gtagggcagtgc
ttgtctccccag CGAGTGGCA
Lys910
1.7
19
1512
Fig.
2 summarizes the genomic organization of human apoER2
and compares it with those of human LDLR and VLDLR. Exon 1 extends 236 bp upstream of the initial methionine codon (see below) and contains
the signal sequence. Cysteine-rich repeats 1-3 and 7 of the
ligand-binding domain are each encoded by individual exons. The other
repeats are all contained in a single exon. Although the ligand-binding
domain of human apoER2 consists of seven cysteine-rich repeats like
that of LDLR, the exon/intron organization of this domain is much more
closely related to that of the human VLDLR gene, which contains an
extra exon encoding an additional cysteine-rich repeat. Like the human
LDLR and VLDLR genes, the growth factor repeats in the apoER2 gene are
each encoded by individual exons. Exons 9-13 encode the nonrepetitive
sequences that are shared among LDLR, VLDLR, and epidermal growth
factor precursor. Like the LDLR and VLDLR genes, the
O-linked sugar domain of the apoER2 gene is encoded by a
single exon of 225 bp. The transmembrane domain is interrupted by a
single intron. Exon 17 contains the C-terminal half of the
transmembrane domain and the first 44 amino acids of the cytoplasmic
domain. Unlike LDLR and VLDLR, the cytoplasmic domain of apoER2
contains an insertion sequence of 59 amino acids. This insertion
sequence is encoded by a single exon of 177 bp (exon 18). The last exon
encodes the remaining 12 amino acids and the 3-untranslated region of
the mRNA.
Chromosome Location of the Human ApoER2 Gene
To localize the human apoER2 gene to a specific chromosome, two techniques were used. First, a PCR strategy was used with the National Institute of General Medical Sciences human/rodent somatic cell hybrid mapping panel 2. A set of PCR primers were designed specifically to detect apoER2 human genomic DNA and not to amplify the rodent (mouse or hamster) apoER2 gene. Analysis of DNA from human/rodent hybrid cells by human sequence-specific PCR revealed that the gene is located on chromosome 1 (data not shown). The apoER2 gene has 0% discordance only with chromosome 1, and we therefore conclude that the apoER2 gene is located within human chromosome 1.
The human apoER2 gene was more precisely located on chromosome 1 using
color-labeled fluorescent in situ hybridization analyses. The entire region of the human apoER2 cDNA was nick-translated with
biotin-16-dUTP and visualized. We consistently observed hybridization signals on chromosome 1 at band p34 (Fig. 3). Thus, the
human apoER2 gene is on a chromosome different from those of LDLR and VLDLR: the genes for LDLR (32) and VLDLR (22, 33) are located on
chromosomes 19p13 and 9p24, respectively.
Splicing Variants
In the course of cloning a cDNA
encoding human apoER2 (pNR1), we obtained a novel cDNA (designated
pNR2) with a deletion, which was presumably derived from an alternative
splicing of the pre-mRNA. Nucleotide sequencing of this cDNA
revealed that the cDNA encodes a variant apoER2 lacking repeats
4-7 in the ligand-binding domain as illustrated in Fig.
4A. This deletion corresponds to the skipping
of exons 5 and 6 during RNA processing. The variant receptor lacking
binding repeats 4-7 was designated apoER24-7. To test whether this
variant indeed recognizes apoE, pNR2 was introduced into LDLR-deficient
Chinese hamster ovary cells (26), and ligand binding was measured using
125I-labeled
-VLDL. As shown in Fig. 4B,
cells expressing the variant receptor bound apoE-rich
-VLDL with
high affinity: the calculated Kd values of
apoER2
4-7 (0.86 µg/ml) and apoER2 (0.86 µg/ml) for
-VLDL
were exactly the same (Fig. 4B, inset). This result indicates that the deletion of binding repeats 4-7 in the ligand-binding domain of apoER2 has essentially no effect on
-VLDL binding.
The recently identified chicken protein LR8B, the chicken homologue of
apoER2, contains an 8-fold cysteine-rich repeat in the ligand-binding
domain (15). Together with our demonstration of a variant lacking
repeats 4-7, this suggests the presence of multiple variants with
different numbers of cysteine-rich repeats in the ligand-binding domain
of the receptor. To test this possibility, RT-PCR was carried out using
poly(A) RNA from human brain and placenta and a pair of oligonucleotide
primers that flank the region corresponding to the ligand-binding
domain of the human receptor. Under standard conditions, we obtained
multiple PCR products of unexpected length, presumably because of
pre-PCR mispriming. To prevent possible pre-PCR mispriming (34), a hot
start PCR was carried out. The RT-PCR RNA gave three major bands with
581, 704, and 1091 nucleotides (Fig. 5A). The
three PCR products were subcloned into T-vectors and sequenced. The
sequences of the 581- and 1091-nucleotide fragments fully matched those
of the corresponding regions in pNR2 and pNR1, respectively. The
704-nucleotide fragment lacked 387 nucleotides corresponding to repeats
4-6 of the ligand-binding domain. This result agrees with our
isolation of two cDNAs, which together indicate that human tissues
express multiple forms of the receptor. We did not detect a variant
with an 8-fold cysteine-rich repeat like chicken LR8B in the human
tissues.
Comparison of the human apoER2 cDNA with chicken LR8B also revealed that the chicken homologue lacks the 59-amino acid insertion sequence in the cytoplasmic domain. To analyze the region corresponding to the cytoplasmic domain of the human receptor, RT-PCR was carried out using a pair of specific primers that span the relevant region. As shown in Fig. 5B, the variant lacking the insertion sequence was also expressed in human tissues. Although the function of the cytoplasmic insertion sequence of apoER2 is currently unknown, it may play a unique role in mammals: LR8B transcripts with the 59-amino acid insertion sequence in the cytoplasmic domain are not detected in chicken brain.2 Further functional analysis is required to elucidate the exact role of this domain in mammals.
Characteristics of the 5The transcription start site was determined by primer
extension analysis using poly(A) RNA from human placenta and brain. For
primer extension analysis, we used a 21-mer oligonucleotide (oligonucleotide 180R) labeled with 32P at the 5-end. In
the primer extension analysis, we detected a major band corresponding
to the position 236 bp upstream of the AUG translation initiator codon
(Fig. 6A). To confirm these results, RT-PCR
was carried out using four pairs of PCR primers. The first cDNA
synthesis was primed with random hexamers and then amplified with three
sets of oligonucleotides, an antisense primer (oligonucleotide R) and
three sense primers (oligonucleotides A-C) (Fig. 6B). In
combinations of oligonucleotide R with oligonucleotides B and C,
amplified cDNA fragments of the expected sizes (198 and 286 bp)
were detected, but no amplification occurred with oligonucleotides R
and A (Fig. 6B). These data indicate that the most upstream transcription initiation site is located between nucleotides
73 and
+69. Based on the primer extension analysis, the transcription site was
defined as the G 236 nucleotides upstream of the initiator methionine.
Fig. 7 shows the nucleotide sequence of the 5-flanking
region of human apoER2. Neither a typical TATA box sequence (35), its
homologue, nor a typical CCAAT box (36) was found in the 5
-flanking
region. Potential sites for Sp1 (37, 38) are present at nucleotides
331,
276, and
32. The flanking region contains DNA motifs for
AP-2 (39, 40) and a potential site for the GC factor, a negative
regulator of the epidermal growth factor gene (41). There is also a
potential site for PEA3 (olyomavirus nhancer
ctivator ), a brain-specific transcriptional
activator (42, 43).
Functional Analysis of the 5
The apoER2
mRNA is predominantly expressed in human brain and placenta and is
undetectable in other tissues including liver, heart, skeletal muscle,
and spleen (10). It is also expressed in rat adrenal pheochromocytoma
PC12 cells and is increased by treatment of the cells with NGF (10).
The maximal accumulation of apoER2 mRNA in PC12 cells by NGF occurs
as early as 3 h,3 suggesting that
transcription is induced rapidly upon the stimulation. To confirm that
the 5-flanking region actually confers promoter activity and to
identify the element(s) that respond to NGF, different lengths of
5
-upstream region of the first exon were fused to the luciferase gene.
Following transfection into neuronal PC12 and non-neuronal HepG2 cells,
the reporter constructs pLAER437 and pLAER316, containing nucleotides
437 to +8 and nucleotides
316 to +8, respectively, produced
significant luciferase activities in both cell lines compared with a
promoterless vector (pPGV-B) (Fig. 8). Consistent with
the accumulation of the mRNA in PC12 cells by NGF, NGF treatment
caused a 2-3-fold induction of luciferase activities in PC12 cells
transfected with pLAER437 and pLAER316, suggesting that the promoter
contains elements inducible by NGF. Although the reporter construct
pLAER148, containing nucleotides
148 to +8, produced apparently no
luciferase activity in HepG2 cells, it was active in PC12 cells, but
the NGF inducibility was lost. These results indicate that the proximal
316 bp relative to the transcription site contain the minimal promoter
that functions in both PC12 and HepG2 cells. Our data also indicate
that the region containing nucleotides
316 to
148 is required for
NGF inducibility in PC12 cells. This region contains potential binding sites for Sp1, AP-2, and PEA3 (Fig. 7). Of particular interest are the
AP-2 and PEA3 sites. AP-2 is a factor known to mediate induction by
12-O-tetradecanoylphorbol-13-acetate and cAMP (40), and the
PEA3 motif is known to be a growth factor- and
12-O-tetradecanoylphorbol-13-acetate-responsive element
(42). Whether the above elements are involved in transcriptional regulation of the apoER2 gene is currently under investigation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D86389[GenBank], D86390[GenBank], D86391[GenBank], D86392[GenBank], D86393[GenBank], D86394[GenBank], D86395[GenBank], D86396[GenBank], D86397[GenBank], D86398[GenBank], D86399[GenBank], D86400[GenBank], D86401[GenBank], D86402[GenBank], D86403[GenBank], D86404[GenBank], D86405[GenBank], D86406[GenBank], D86407[GenBank].
We thank Drs. Mike Brown and Joe Goldstein for helpful advice and discussions, Dr. Monty Krieger for ldlA-7 cells, Dr. Ian Gleadall for critical reading of the manuscript, and Kyoko Ogamo Karasawa and Nami Suzuki for secretarial assistance.