©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Apolipoprotein E Receptor 2
A NOVEL LIPOPROTEIN RECEPTOR OF THE LOW DENSITY LIPOPROTEIN RECEPTOR FAMILY PREDOMINANTLY EXPRESSED IN BRAIN (*)

(Received for publication, August 23, 1995; and in revised form, January 16, 1996)

Dong-Ho Kim (1) Hiroaki Iijima (1) Kaoru Goto (2) Juro Sakai (1)(§) Hirofumi Ishii (1) Hyoun-Ju Kim (1) Hiroyuki Suzuki (1) Hisatake Kondo (2) Shigeru Saeki (1) Tokuo Yamamoto (1)(¶)

From the  (1)Tohoku University Gene Research Center, Sendai 981 and the (2)Department of Anatomy, Tohoku University School of Medicine, Sendai 980, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Isolation and characterization of a human cDNA demonstrated a novel lipoprotein receptor designated apolipoprotein E receptor 2 (apoER2). The new receptor consists of five functional domains resembling the low density lipoprotein (LDL) and very low density lipoprotein (VLDL) receptors. LDL receptor deficient Chinese hamster ovary cells expressing human apoER2 bound apoE rich beta-migrating VLDL with high affinity and internalized. LDL was bound with much lower affinity to these cells. The 4.5- and 8.5-kb mRNAs for the receptor were most highly expressed in human brain and placenta. In rabbit tissues, multiple species of the mRNA with 4, 4.5, 5.5, 8.5, and 11 kb were detected most intensely in brain and testis and, to a much lesser extent, in ovary, but were undetectable in other tissues. In rat adrenal pheochromocytoma PC12 cells, the receptor mRNA was induced by treatment of the cells with nerve growth factor. The receptor transcripts were detectable most intensely in the cerebellar cortex, choroid plexus, ependyma, hippocampus, olfactory bulb and, to a much lesser extent, in the cerebral cortex as revealed by in situ hybridization histochemistry. In the cerebellar cortex, the receptor transcripts were densely deposited in Purkinje cell somata.


INTRODUCTION

Receptor-mediated endocytosis of plasma lipoproteins plays an important role in the metabolism of cholesterol and triglyceride in the body. The low density lipoprotein (LDL) (^1)receptor, one of the best characterized cell surface receptors, mediates cholesterol homeostasis in the body(1) . The LDL receptor binds apolipoprotein B-100 containing LDL and apolipoprotein E (apoE)-containing lipoproteins, whereas the recently found very low density lipoprotein (VLDL) receptor binds only apoE-containing lipoproteins(2, 3) . Both the LDL receptor (4, 5, 6, 7) and VLDL receptor(2, 8, 9, 10, 11, 12, 13, 14) consist of five functional domains: (i) an amino-terminal ligand binding domain composed of multiple cysteine-rich repeats; (ii) an epidermal growth factor (EGF) precursor homology domain, which mediates the acid-dependent dissociation of the ligands from the LDL receptor(15) ; (iii) an O-linked sugar domain; (iv) a transmembrane domain; and (v) a cytoplasmic domain with a coated pit targeting signal(16) . Genetic deficiencies of the LDL receptor give rise to familial hypercholesterolemia, one of the most common genetic diseases in humans(17) . Mutations in the chicken VLDL receptor gene lead to the failure to produce offspring(13, 18) .

Lipoprotein metabolism in the central nervous system (CNS) has been poorly understood, despite the importance of lipids in some specialized neural membranes, such as myelin. Most of lipids in the CNS are actively synthesized in the CNS itself and deposited in large amounts during the early phase of development(19, 20) . The rate of cholesterol and fatty acid synthesis in the brain is high during the myelinating period and declines thereafter(19, 20) . Although most of lipids in the brain are believed to be synthesized within the brain itself, small amounts of cholesterol (21) and fatty acid (22) are taken up by the brain throughout life. These observations have suggested the presence of a mechanism for exchange of lipids between the CNS and the general circulation.

ApoE, a major cholesterol and triglyceride-carrying protein in plasma, is secreted by hepatic and extrahepatic cells and mediates high-affinity binding of apoE-containing lipoproteins to the LDL and VLDL receptors. In the brain, significantly high levels of apoE are synthesized and secreted by astrocytes(23) . ApoE phospholipid discoidal particles or apoE-enriched high density lipoprotein (HDL) found in cerebrospinal fluid are thought to be taken up by brain cells via receptor mediated endocytosis, for which receptors for apoE, including the LDL and VLDL receptors, probably play a key role in the CNS.

LDL receptor mRNA is expressed at particularly high levels in sensory ganglia, sensory nuclei, and motor-related nuclei in rabbit brain(24) . Hofmann et al.(25) have shown that the levels of the LDL receptor mRNA did not decline when myelination of the CNS was completed, indicating that the brain required LDL receptors even in adult life. VLDL receptor mRNA is also expressed in the mammalian brain(2, 8, 10, 12, 14, 26) , but little is known of its role in the CNS.

As an initial approach to understand more about the apoE-lipoprotein metabolism in the CNS, we have isolated a cDNA encoding a novel apoE receptor predominantly expressed in the brain. In the current paper, we describe the structure, ligand specificity, and expression of a novel apoE-specific lipoprotein receptor designated apoE receptor 2 (apoER2).


EXPERIMENTAL PROCEDURES

General Methods

Standard molecular biology techniques were carried out essentially as described by Sambrook et al.(27) . Nucleotide sequencing was performed by the dideoxy-chain termination method (28) using M13 primers, T3 and T7, or specific internal primers. Sequence reactions were carried out using Taq DNA polymerase with fluorescently labeled nucleotides on an Applied Biosystems model 373A DNA sequencer. To analyze RNA in human tissues, commercially available Northern blots (Clontech) were hybridized with P-labeled probes primed with random hexanucleotides(29) . The probes used to analyze the Northern blots included the human apoER2 cDNA extended from nucleotides 388 to 1159, the human LDL receptor cDNA probe extended from nucleotides 54 to 718(4) , and the human VLDL receptor cDNA probe extended from nucleotides 1000 to 1794(9) .

cDNA Cloning

A cDNA library was constructed in an Okayama-Berg vector (30) using poly(A) RNA from human placenta and screened with a mixture of degenerate oligonucleotides corresponding to a coated pit signal (FDNPVY) highly conserved between the LDL and VLDL receptors(2) : 5`-TT(C/T)GA(C/T)AA(C/T)CC(A/C/G/T)GT(A/C/G/T)TA-3`. Positive clones that hybridized with the oligonucleotides were then reprobed with the LDL and VLDL receptor probes to eliminate LDL and VLDL receptor cDNAs. Screening of 5 times 10^5 clones, we obtained one positive clone (pNR1) that hybridized with the oligonucleotide probes alone.

Isolation of Transfected Cell Lines

For the lipoprotein receptor assay, pNR1 was cotransfected with pSV2-Neo (31) into ldlA-7 cells(32) , a mutant Chinese hamster ovary (CHO) cell line lacking LDL receptor, according to the transfection protocol described by Chen and Okayama(33) . After the transfection, the cells were placed in selective medium containing 700 µg/ml of G418. ldlA-7 cells expressing human LDL receptor were isolated as described previously(2) .

Lipoprotein Preparation and Lipoprotein Binding Assays

Human LDL (d = 1.019-1.063 g/ml), VLDL (d < 1.006 g/ml), and lipoprotein-deficient serum (d > 1.21 g/ml) were prepared as described previously(34) . Rabbit beta-VLDL (d < 1.006 g/ml) was prepared from the plasma of 1% cholesterol-fed animals(35) . I-Labeled lipoproteins were prepared as described previously(36) . Fluorescently labeled beta-VLDL was prepared using 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate as described(2) . The receptor-mediated endocytosis of beta-VLDL by the transfected cells was visualized by using fluorescently labeled beta-VLDL at 37 °C. Binding and internalization of I-labeled LDL, VLDL, and beta-VLDL by the transfected cells were assayed according to the procedure as described (36) except that dextran sulfate was replaced by suramin to release bound lipoproteins(15) .

Cell Culture

Rat PC12 pheochromocytoma cells were grown in Dulbecco's modified Eagle's medium containing 5% horse serum and 5% fetal bovine serum. To differentiate PC12 cells, the cells were treated with NGF (Life Technologies, Inc.) for 72 h. Rat KEG1 glioma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

In Situ Hybridization Histochemistry

In situ hybridization procedures were carried out as described(37) . Cryostat sections (30 µm) from adult male rats were hybridized either with S-labeled rat LDL receptor cDNA (extended form 1092-2095)(7) , rat VLDL receptor cDNA (extended form 1690-2756) (14) , or rat apoER2 cDNA (extended form 2062-3070). (^2)After exposure to Hyperfilm-betamax (Amersham) for 3 weeks, the sections were dipped in Kodak NTB2 emulsion and exposed for 3 months.


RESULTS AND DISCUSSION

Isolation of a cDNA Encoding a Novel Lipoprotein Receptor

Using a mixture of degenerate oligonucleotides that correspond to the highly conserved coated pit signal, FDNPVY, in the cytoplasmic domains of the LDL and VLDL receptors(2, 9) , we obtained one positive clone (designated pNR1) that encodes a novel protein highly similar to the LDL and VLDL receptors (Fig. 1). To verify that the cDNA encodes a lipoprotein receptor, the cDNA was introduced into LDL receptor-deficient ldlA-7 cells by cotransfection with pSV2-Neo. G418 resistant cells were isolated and the lipoprotein binding activities were visualized by using fluorescent beta-VLDL. Fluorescent beta-VLDL was actively taken up by the transfected cells, but there was no accumulation of fluorescent beta-VLDL in control cells transfected with pSV2-Neo alone (Fig. 2), indicating that the cDNA encodes a lipoprotein receptor. Based on the ligand specificity of the receptor (see below), we designated this new receptor apoER2.


Figure 1: Structure of the human apoER2 cDNA. The deduced amino acid sequence of human apoER2. Amino acids are numbered on the left side; residue 1 is the glycine believed to constitute the NH(2) terminus of the mature protein; negative numbers refer to the cleaved signal sequence (boxed in black, NH(2) terminus). Cysteine residues are circled. Two potential sites of N-linked glycosylation (Asn-X-Ser or Asn-X-Thr) are boxed. Serine and threonine residues in a region that corresponds to the O-linked sugar domain of the receptor are underlined. The 22-residue transmembrane segment located toward the COOH terminus of the protein is boxed in black. The FDNPXY sequence (32) required for clustering of the LDL receptor in coated pits is indicated by a dotted underline. The stop codon is indicated by an asterisk. Potential polyadenylation sites are indicated by overlines and underlines.




Figure 2: Visualization of receptor-mediated uptake of fluorescent beta-VLDL by CHO cells expressing human apoER2. Human apoER2 cDNA (pNR1) (A) or human LDL receptor cDNA (pLDLR2) (B) were introduced into LDL receptor-deficient ldlA-7 cells together with pSV2-Neo. Control cells (C) were transfected with pSV2-Neo alone. G418-resistant cells were incubated with fluorescent beta-VLDL for 4 h at 37 °C. Magnification: times 241.1.



Deduced Amino Acid Sequence of the Primary Translation Product

The insert of the cDNA includes a 2889-bp open reading frame encoding a protein of 963 amino acids with a calculated M(r) of 105,714 (Fig. 1). The NH(2)-terminal 41 amino acid sequence (residues -41 to -1) has the characteristics of a classical signal sequence. The amino acid sequence following the 41st residue (Gln) contains a cysteine-rich sequence similar to that of the NH(2)-terminal of the mature human VLDL receptor (Fig. 3). Therefore we tentatively assigned the 42nd residue (Gly^1 in Fig. 1and Fig. 3) as the NH(2) terminus of the mature protein. The mature human apoER2 would then consist of 922 amino acids with a calculated M(r) of 101,440.


Figure 3: Comparison of the amino acids in human apoER2 with those of human LDL and VLDL receptors. A, the amino acids in the five functional domains of human apoER2 were compared with those in the human LDL receptor (LDLR) (4) and VLDL receptor (VLDLR)(9) . Amino acids are numbered on the left. Identical amino acids are boxed. The percentage amino acid identities within each domain of human apoER2 versus human LDL receptor and human apoER2 versus human VLDL receptor are listed on the right. B, schematic representation of human apoER2, LDL receptor (LDLR) and VLDL receptor (VLDLR). The cysteine-rich repeats in the ligand binding domains are assigned the Roman numerals I-VIII. The linkers separating cysteine-rich repeats in the ligand binding domain are indicated by filled boxes. The cysteine-rich repeats in the EGF precursor homology domains are lettered A-C. The insertion sequence in the cytoplasmic domain of human apoER2 is stippled.



Comparison of the Amino Acids in Human ApoER2 with Those in the LDL and VLDL Receptors

The deduced amino acid sequence of the cDNA revealed that human apoER2 consists of five functional domains resembling the LDL and VLDL receptors. The NH(2)-terminal 294 amino acid sequence of human apoER2 is composed of a 40-amino acid sequence repeated seven times, with characteristics similar to those of the cysteine-rich repeats in the ligand binding domains of the LDL receptor (4, 5, 6, 7) and VLDL receptor(2, 8, 9, 10, 11, 12, 13, 14) (Fig. 3). Approximately 50% of the amino acids in each repeat of the ligand binding domain of human apoER2 are identical to those in the human LDL and VLDL receptors. Although human apoER2 and the LDL receptor contain the same number of cysteine-rich repeats, the ligand binding domain structure of human apoER2 is much more closely related to that of the VLDL receptor, despite the presence of an extra repeat in the latter (Fig. 3). A short linker sequence is present between repeats 5 and 6 of human apoER2 and the VLDL receptor, whereas in the LDL receptor it is located between repeats 4 and 5.

The EGF precursor homology domain of the LDL receptor consists of three cysteine-rich repeats (known as growth factor repeats) and mediates the acid-dependent dissociation of the ligand(15) . This domain in human apoER2 also has three growth factor repeats and approximately 55% of the amino acids are identical to those in the human LDL and VLDL receptors. This high degree of amino acid identity suggests that apoER2 also dissociates from its ligands in the endosomes.

The O-linked sugar domain human apoER2 contains 89 amino acid residues, including 36 serine and threonine residues. The amino acid identity between the O-linked sugar domains of human apoER2 and the human VLDL receptor or LDL receptor is only 21 and 27%, respectively. The amino acids in the transmembrane domains of the human VLDL and LDL receptors are also poorly conserved in human apoER2: only 35% of the amino acids in this domain of apoE receptor are identical to those in the human VLDL and LDL receptors.

The cytoplasmic domain of human apoER2 consists of 115 amino acids, whereas in the LDL and VLDL receptors there are only 50 and 54 amino acids, respectively. The cytoplasmic domain of the LDL receptor contains a coated pit signal (16) and a basolateral sorting signal (38) . The NH(2)-terminal 25 amino acids of the cytoplasmic domain of human apoER2 are closely similar to those surrounding the coated pit signal of the LDL receptor and the NH(2)-terminal half of the cytoplasmic domain of the VLDL receptor(2) , suggesting that apoER2 is also clustered in coated pits and mediates the internalization of its ligands. Amino acid comparison of the cytoplasmic domains of the three human lipoprotein receptors revealed a unique insertion sequence of 59 amino acids in the cytoplasmic domain of human apoER2. This insertion may constitute a signal required for the specific localization of the receptor in some specialized region of the neural cell membrane, since the basolateral targeting signal of the LDL receptor is also located between the coated pit signal and COOH terminus of the LDL receptor(38) .

Lipoprotein Specificity

The ligand specificity of human apoER2 expressed in ldlA-7 cells was compared with that of cells expressing the human LDL receptor using I-labeled LDL, VLDL, and beta-VLDL. The cells expressing human apoER2 bound apoE-rich beta-VLDL with high affinity, but LDL and VLDL were bound with much lower affinity (Fig. 4A). Cells expressing human LDL receptor bound I-labeled LDL, VLDL, and beta-VLDL with high affinity (Fig. 4B). Like the LDL receptor expressing cells, the I-beta-VLDL bound to human apoER2 was internalized (Fig. 4, C and D).


Figure 4: Ligand specificity of human apoER2 expressed in CHO cells. Surface binding (upper panels) and internalization (lower panels) of I-labeled LDL, VLDL, and beta-VLDL in CHO cells expressing human apoER2 (A, C) or human LDL receptor (B, D). For surface binding assay, CHO cells transfected with a plasmid encoding human apoER2 (pNR1) or human LDL receptor (pLDLR2) were incubated for 2 h at 4 °C with the indicated concentrations of I-LDL (323 cpm/ng), I-VLDL (114 cpm/ng), or I-beta-VLDL (184 cpm/ng). For the internalization assay, transfected cells were incubated for 3 h at 37 °C with the indicated concentrations of I-labeled lipoproteins, after which the values for internalized I-labeled lipoproteins were determined. Specific values for a given ligand were calculated by subtracting the values obtained with pSV2-Neo transfected cells. Each value represents the average of two incubations.



Although human apoER2 consists of seven cysteine-rich repeats highly similar to those of the LDL receptor, it binds only apoE-rich beta-VLDL. A key structural difference in the ligand binding domains of the two receptors is the position of the linker sequence (see Fig. 3). Russell et al.(39) have shown that deletion of the linker sequence in the LDL receptor drastically reduced LDL binding, but had no effect on the binding of beta-VLDL. Therefore, it is likely that the strategic location of the linker sequence between the cysteine-rich repeats in the two lipoprotein receptors contributes to their ligand specificity.

Tissue-specific Expression

Northern blot analysis of RNA from various human tissues revealed hybridization of the human apoER2 probe to major transcripts of 4.5- and 8.5-kb in brain (Fig. 5A). The 4.5- and 8.5-kb mRNAs are also detectable in placenta, where the 8.5-kb mRNA is a minor transcript. The 8.5 kb transcript may be a consequence of alternative splicing or alternative polyadenylation. The mRNA was undetectable in heart, lung, liver, skeletal muscle, kidney, or pancreas. This pattern of tissue distribution and relative abundance of apoER2 mRNA are completely different from those of the LDL and VLDL receptors: the VLDL receptor mRNA is most highly expressed in heart and muscle (Fig. 5B), whereas the LDL receptor mRNA is expressed in various tissues including liver (Fig. 5C).


Figure 5: Northern blot analysis of apoER2 mRNA. 2 µg of poly(A) RNA from the indicated tissues was probed with human apoER2 (A), VLDL receptor (B), or LDL receptor (C) cDNA probes. The filters were exposed to Kodak XAR-5 film with an intensifying screen for 72 h at -70 °C. The same samples in A were subsequently hybridized with a control probe for human cyclophilin (45) and exposed to Kodak XAR-5 film with an intensifying screen for 12 h at -70 °C. Northern blot analysis of apoER2 mRNA in various rabbit tissues (D). Total RNA (15 µg) prepared from the indicated rabbit tissue was hybridized with the human apoER2 probe and exposed to Kodak XAR-5 film with an intensifying screen for 72 h at -70 °C. The same filter was subsequently hybridized with a control probe for human cyclophilin (45) and exposed to Kodak XAR-5 film with an intensifying screen for 12 h at -70 °C. Expression of apoER2 mRNA in rat PC12 and KEG1 cells (E). Rat PC12 and KEG1 cells were cultured as described under ``Experimental Procedures.'' To induce differentiation of PC12 cells, the cells were treated NGF (50 ng/ml) for 3 days. Total cellular RNA (15 µg) was hybridized with the rat apoER2 probe and exposed to Kodak XAR-5 film with an intensifying screen for 60 h at -70 °C. The same filter was subsequently hybridized with a control probe for human cyclophilin (45) and exposed to Kodak XAR-5 film with an intensifying screen for 10 h at -70 °C.



Fig. 5D shows a blot hybridization of total RNAs from various rabbit tissues probed with the human apoER2 cDNA. Hybridization to multiple transcripts with 4, 4.5, 5.5, 8.5, and 11 kb were detectable most intensely in brain and testis and, to a much lesser extent, in ovary, but undetectable in other tissues. The 8.5-kb species is the major transcript in cerebrum, cerebellum, brain stem, and ovary, whereas the 4- and 4.5-kb species were most abundant in testis. The 5.5- and 8.5-kb mRNAs for rat apoER2 were found in rat adrenal pheochromocytoma PC12 and glioma KEG1 cells (Fig. 5E). Treatment of PC12 cells with NGF caused 2.3-fold increase in the levels of the transcripts, suggesting that transcription of the apoER2 gene in PC12 cells is stimulated by NGF.

In situ hybridization analysis of adult rat brain showed that the apoER2 transcripts were detectable most intensely in the cerebellar cortex, choroid plexus, ependyma, hippocampus, olfactory bulb and, to a much lesser extent, in the cerebral cortex (Fig. 6A). The transcripts for LDL and VLDL receptors (Fig. 6, B and C) were also detectable in the cerebellar cortex and hippocampus, but no significant signals were detected in the choroid plexus, ependyma, or olfactory bulb, suggesting that the role of apoER2 in the CNS is different from those of the LDL and VLDL receptors. In control experiments, brain sections were hybridized with the plasmid vector of an appropriate length, or some were pretreated with RNase before hybridization. In either case, no significant signals were detectable in any brain regions (data not shown).


Figure 6: In situ hybridization of apoER2 transcripts in the adult rat brain. Autoradiographic film images showing the patterns of hybridization with S-labeled rat apoER2 (A), rat LDL receptor (B), and rat VLDL receptor (C) probes to parasagittal sections through the caudate putamen. In A, note dense hybridization with the apoER2 probe (hybridization signals appear white) in the cerebellar cortex (Cb), choroid plexus and ependyma (arrowheads), hippocampus (Hip), and olfactory bulb (OB) and moderate signals in the cerebral cortex (Cx). The cerebellar cortex and hippocampus are also labeled with the LDL receptor (B) and VLDL receptor (C) probes. Th, thalamus. Scale: 1 mm. D and E, dark field photomicrographs at higher magnification showing the patterns of hybridization with S-labeled rat apoER2 (D), rat LDL receptor (E), and rat VLDL receptor (F) probes to the cerebellum. Note intense signals in the Purkinje cells (arrowheads) hybridized with the apoER2 (D) and LDL receptor (E) probes, and moderate signals in the Purkinje cell layer hybridized with the VLDL receptor probe (F). g, granular layer; m, molecular layer; and w, white matter. Magnification: times 33.7.



In the cerebellar cortex, apoER2 and LDL receptor transcripts were densely deposited in the Purkinje cell somata (Fig. 6, D and E). A similar pattern was detected by the VLDL receptor probe, but it could not be determined whether the signals were in the Purkinje cells or in the Bergmann's glial cells surrounding the Purkinje cells (Fig. 6F).

ApoER2 is a new member of the LDL receptor super family that includes the VLDL receptor(2, 8, 9, 10, 11, 12, 13, 14) , LDL receptor-related protein (LRP)(40) , a kidney glycoprotein termed gp330/megalin(41, 42) , a LRP-like molecule in Caenorhabditis elegans(43) , a recently identified Drosophila vitellogenin receptor (44) and the LDL receptor itself(4, 5, 6, 7) . The structure of apoER2 is most closely related to that of the LDL receptor. The predominant expression of apoER2 mRNA in brain is different from those of the LDL and VLDL receptors, LRP and GP330/megalin: LRP is expressed in various tissues, including liver, intestine, lung, and brain(40) ; whereas GP330/megalin is abundant in kidney(42) . The abundant expression of the mRNA for apoER2 in the brain suggests that it plays a key role in the uptake of apoE phospholipid discoidal particles or apoE-enriched HDL in the CNS. Although the exact nature of this receptor remains to be elucidated, this finding will promote studies on the metabolism and regulation of apoE in the CNS.


FOOTNOTES

*
This research was supported in part by research grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) D50678[GenBank].

§
Present address: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235.

To whom correspondence and reprint requests should be addressed: Tohoku University Gene Research Center, 1-1 Tsutsumidori-Amamiya, Aoba, Sendai 981, Japan. Tel.: 81-22-271-2815; Fax: 81-22-263-9295.

(^1)
The abbreviations used are: LDL, low density lipoprotein; apoE, apolipoprotein E; apoER2, apolipoprotein E receptor 2; CHO cells, Chinese hamster ovary cells; CNS, central nervous system; HDL; high density lipoprotein; EGF, epidermal growth factor; LRP, LDL receptor related protein; NGF, nerve growth factor; VLDL, very low density lipoprotein; beta-VLDL, beta-migrating very low density lipoprotein; kb, kilobase(s).

(^2)
D.-H. Kim and T. Yamamoto, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Mike Brown and Joe Goldstein for helpful advice and discussions, Dr. Monty Krieger for ldlA-7 cell and Dr. Ian Gleadall for critical reading of the manuscript.


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