(Received for publication, August 23, 1995; and in revised form, January 16, 1996)
From the
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 -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.
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) ()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).
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 terminus of the
mature protein; negative numbers refer to the cleaved signal
sequence (boxed in black, NH
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 -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
-VLDL for 4 h at 37 °C.
Magnification:
241.1.
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.
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-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
-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) .
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
-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-
-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 -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
-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.
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:
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D50678[GenBank].