Rapid Communication J. Biochem.124, 1072-1076 (1998)

A New Low Density Related , LRP5, Is Expressed in Hepatocytes and Adrenal Cortex, and Recognizes E 1

Dong-Ho Kim,* Yousuke Inagaki, * Takashi Suzuki,t Ryoichi X. Ioka, * Shiroh Z. Yoshioka, * Kenta Magoori,* Man-Jong Kang,* Yuko Cho,* Akira Z. Nakano,* Qiong Liu,* Takahiro Fujino,* Hiroyuki Suzuki,* Hironobu Sasano,•õand Tokuo T. Yamamoto*,2

*Tohoku University Research Center , Sendai 981-8555; and •õDepartment of Pathology, School of Medicine, Tohoku University, Sendai 981-8575

Received for publication, September 24, 1998

The isolation and characterization of rabbit and human cDNAs revealed a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1,616- type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibited increased binding of (apoE)-enriched ,Q-migrating very low density lipoprotein. Northern blotting and in situ hybridization revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA was increased in the liver and accumulated in -laden foam cells of atherosclerotic lesions.

Key words: apoE, LDL receptor family, LDL receptor-related protein, lipoprotein.

Apolipoprotein E (apoE) is a major component of plasma previous studies, we characterized two LDLR-related including remnants, intermediate (LRPs) termed LRP3 (3) and LRP4 (4). Although density lipoprotein (IDL), and very low density lipoprotein these proteins contain several ligand binding repeats, they (VLDL). These apoE-containing lipoproteins are recog fail to bind ƒÀ-migrating VLDL (ƒÀ-VLDL), an apoE-rich nized by several receptors including apoE receptor 2 lipoprotein induced by cholesterol feeding. (apoER2), low density lipoprotein (LDL) receptor (LDLR), In the current paper, we report a new LRP, designated as and VLDL receptor (VLDLR), and are carried into cells via LRP5, that is expressed in the liver and adrenal gland, and receptor-mediated endocytosis (reviewed in Ref. 1). recognizes apoE. ApoER2 and VLDLR consist of five functional domains Standard molecular biology techniques were carried out resembling LDLR (2), and bind only apoE-containing essentially as described by Sambrook et al. (5). To analyze lipoproteins, whereas LDLR recognizes both apolipoprotein RNA in human tissues, commercially available Northern B-100 (apoB-100) and apoE. LDLR is expressed in various blots (Clontech) were used for Northern blot analysis. tissues including the liver, while VLDLR and apoER2 are cDNA libraries were constructed in pBluescript vector almost completely absent in the liver. Although LDLR using poly(A) RNA and a cDNA synthesis kit from Phar plays a key role in the hepatic clearance of cholesterol- macia. carrying LDL, the presence of other apoE-specific recep Tissue specimens were obtained from homozygous tors has been suggested on the use of LDLR lacking WHHL rabbits (male, 10-12 months old, n=3) and Japa Watanabe heritable hyperlipidemic rabbits (WHHL rab nese white rabbits (male, 10-12 months old, n=3). In situ bits) (reviewed in Ref. 1). To identify new receptors that hybridization was performed as previously described (6), may play a role in the clearance of apoE-containing lipo using an antisense and a sense oligonucleotide probe syn- proteins, we have been characterizing cDNAs encoding thesized with a 3•Œ biotinylated tail (Brigati tail). The ligand binding motifs common to the LDLR family. In the sequence of the 29-base antisense probe specific to rabbit LRP5 was as follows: 5•Œ-GTGTGCCAGCGCGTGGTGTG- 1 This work was supported by the Japan Society for the Promotion of CCAGCGCTA-3•Œ (corresponding to 4216-4244 of human Science, Grant RFTF97L00803. The sequence data in this article LRP5). have been deposited in the EMBL/GeneBank Data Libraries under An adenovirus (AdLRP5) containing the entire region of accession No. AB017498-017499. human LRP5 cDNA was generated by multiple ligation of 2 To whom correspondence should be addressed. Fax: +81-22-263- 9295. E-mail: [email protected] restriction fragments using an adenovirus expression Abbreviations: apo, apolipoprotein; apoER2, apolipoprotein E recep vector kit (Takara Shuzo). A recombinant adenovirus, tor 2; LDL, low density lipoprotein receptor; LDLR, low density lipo AdlacZ, which contains the modified chicken ƒÀ-actin pro protein receptor; LRP, low density lipoprotein receptor-related pro moter with the cytomegalovirus-immediate early tein; VLDL, very low density lipoprotein; VLDLR, very low density (7) and the ƒÀ-galactosidase gene, was used as a control. lipoprotein receptor; ƒÀ-VLDL, fl-migrating very low density lipopro AdLRP5 (1.0 x 109 plaque-forming units/ml) and AdlacZ tein; WHHL rabbit, Watanabe heritable hyperlipidemic rabbit. (1.5 x 109 plaque-forming units/ml) were incubated with (C) 1998 by The Japanese Biochemical Society. LDLR-deficient CHO cells (ldlA7) in 0.1 ml of Dulbecco's

1072 J. Biochem. LDL Receptor Related Protein 5 1073

Fig. 1. (A) Deduced amino acid sequences of human and rabbit rabbit LRP5 are indicated by dashes. Cysteine residues are circled. LRP5 cDNAs. Amino acid residue numbers are shown on the right. Potential N-linked glycosylation sites are underlined and the YWTD Amino acid residue 1 is the glycine that is believed to constitute the tetrapeptide is doubly underlined. (B) Schematic representation of N-terminus of the mature protein. Negative numbers refer to the LRPs 1-5, apoER2, LDLR, and VLDLR. cleaved sequence (boxed at the N-terminus). The identical residues in

modified essential medium supplemented with 5% FBS in are identical to those of the human protein. 6-well plates. Seventy-two hours after transduction of the During the preparation of this paper, Hey et al. published adenovirus, the cells were subjected to lipoprotein binding the sequences of human and murine LRP5 (10). Amino acid assaying according to the procedure described (8) using comparison revealed that our human LRP5 sequence is fluorescently labeled lipoproteins (9). completely identical to that of Hev et al. (10). A partial eDNA (designated as pLRX1) encoding a new A hydropathy plot of the deduced amino acid sequence of member of the LDLR family, designated as LRP5, was human LRP5 showed the presence of two hydrophobic initially isolated from a WHHL rabbit eDNA library using regions (boxed in Fig. IA): one at the N-terminus and the the entire region of human apoER2 cDNA as a probe. A other at amino acid residues 1363-1385. The former human eDNA (designated as pLRX2) containing the entire corresponds to a classical signal sequence of probably 23 coding region was then isolated using the rabbit eDNA. amino acids in length, while the latter strongly resembles Figure 1A shows the complete amino acid sequence of the transmembrane region of membrane proteins, suggest- human LRP5 and a part of that of rabbit LRP5 deduced ing that LRP5 has a type I transmembrane protein struc from the cDNAs. The human eDNA contains a 4,848 by ture (C-terminus in the cytosol). open reading frame for 1,616 amino acids with a calculated In the putative extracellular region, there are three molecular mass of approximately 180 kDa. The putative copies of the ligand binding repeat (class A motif) of LDLR initial methionine was preceded by an in-frame termina immediately external to the putative membrane-spanning tion codon 36 nucleotides upstream. The rabbit cDNA sequence (Fig. 1B). The majority of the putative extracel encodes 537 amino acids corresponding to amino acids lular region consists of four copies of a cysteine-poor EGF 1026-1552 of human LRP5, of which approximately 91% precursor-type sequence with five copies of the sequence,

Vol. 124, No. 6, 1998 1074 D.-H. Kim et al.

YWTD, and four copies of a cysteine-rich EGF precursor- in Fig. 2A, cells transduced by AdLRP5 exhibited a slight type repeat (class B motif). increase in the uptake of R-VLDL as compared to control In the putative cytoplasmic region (C-terminus), we cells transduced with an equal titer of AdlacZ (Fig. 2B). found neither a typical internalization signal, FXNPXY, There was no uptake of fluorescent LDL or VLDL by cells nor a similar tyrosine-based sequence (1). transduced by either virus (data not shown). When ƒÀ- To determine whether or not LRP5 binds apoE-con VLDL was preincubated with apoE, increased uptake was taining lipoproteins, we generated a replication-defective observed in cells transduced by AdLRP5 (Fig. 2C), but not adenovirus (AdLRP5) containing the entire coding region of in ones transduced by AdlacZ (Fig. 2D). These data suggest human LRP5 cDNA. AdLRP5 and a control adenovirus that LRP5 may play a role in the metabolism of apoE-con (AdlacZ) containing the lacZ gene were individually trans taining lipoproteins. duced into LDLR-deficient CHO cells, and then ligand Northern blot analysis of RNA from various rabbit binding activity was measured using fluorescently labeled tissues revealed hybridization of the rabbit probe to a rabbit R-VLDL, human LDL, and human VLDL. As shown major transcript of 6.6 kb, with the highest expression in

Fig. 2. Adenovirus-mediated expression of LRP5 in CHO cells. Visualization of receptor mediated endocytosis of fluores cent lipoproteins by LDLR-defi cient CHO cells transduced by adenoviruses encoding human LRP5 (AdLRP5) (A, C) or the lacZ gene (AdlacZ) (B, D). Cells were incubated with fluorescent- ly labeled ƒÀ-VLDL (10,ƒÊg/ml) (A, B) or ,ƒÀ-VLDL (10 jig/ml) preincubated with human apoE (isoform E-3, 7.5,ƒÊg/ml) (C, D) for 3 h at 37•Ž. Magnification: X 80.

Fig. 3. Expression of LRP5 transcripts. Ten micro-

grams of total RNA from the indicated rabbit tissues (A) and 2ƒÊg of poly(A) RNA from the indicated human tissues (B) were probed with 32P-labeled rabbit and human LRP5 cDNAs, respectively. The filters were exposed to Kodak XAR-5 film with an intensifying screen at -80 for 40 h. Control hybridization with a rat

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown in the lower portion. Total RNA (10 kg) isolated from the livers of normal and WHHL rabbits was analyzed with 32P-labeled rabbit LRP5 cDNA (C). Autoradiography was performed at -80•Ž for 40 h with an intensifying screen. The autoradiograph shown is representative of five independent experiments which

gave essentially identical results. Control hybridization with a rat cyclophilin probe (CP) is shown in the lower portion.

J. Biochem. LDL Receptor Related Protein 5 1075

Fig. 4. In situ hybridization analysis of LRP5 transcripts in rabbit tissues. Sections A and C-F were hybridized with an antisense probe to rabbit LRP5. B: The negative control with a sense probe to LRP5. Hybridiza tion signals were visualized, in red. Tissue sections were prepar ed from the liver (A, B), adrenal cortex (C), and adrenal medulla

(D) of a normal rabbit, the ather osclerotic lesion of a WHHL rabbit aorta (E), and the aorta of a normal rabbit (F). LRP5 tran scripts are strongly localized in

parenchymal cells of the liver and adrenocortical parenchymal cells of the adrenal gland. In the ather osclerotic lesion of the WHHL rabbit, marked accumulation of hybridization signals can be seen detected in cholesterol-laden foam cells. Nuclei were made visible by counterstaining with hematoxylin (A-F, Bars=100 ƒÊ m).

lung, heart and adrenal gland, relatively high expression in cholesterol metabolism with cholesterol. In negative con liver, spleen, kidney, small intestine and skeletal muscle, trols for mRNA in situ hybridization using the sense and much lower expression in cerebrum and cerebellum oligonucleotide probe, no significant accumulation of (Fig. 3A). In human tissues, a major transcript of 5.6 kb hybridization signals was detected in the liver (Fig. 4B) or was detected, with the highest expression in heart, liver, adrenal eland (data not shown). pancreas, prostate, ovary, small intestine, colon, thyroid, We also examined the atherosclerotic lesions of WHHL trachea, and adrenal gland, relatively high expression in rabbits by means of mRNA in situ hybridization. As shown placenta, lung, skeletal muscle, kidney, stomach, and spinal in Fig. 4E, hybridization signals are accumulated in chole cord, much lower expression in brain, spleen, thymus, sterol-laden foam cells, whereas no accumulation was testis, lymph node, and bone marrow, and a trace amount in detected with the sense probe (data not shown). In the leukocytes (Fig. 3B). normal areas of WHHL (data not shown) and normal rabbit To determine the effect of cholesterol on LRP5 expres (Fig. 4F) aortae, no significant accumulation of hybridiza sion, we analyzed the transcript levels in the livers of tion signals was detected. These data suggest that LRP5 normal and WHHL rabbits. As shown in Fig. 3C, the may contribute to foam cell formation during the process of hepatic level of LRP5 in WHHL rabbits is 2-3-fold higher atherosclerosis. than that in normal ones, suggesting that LRP5 expression is regulated by the cholesterol level in vivo. REFERENCES To locate cells expressing LRP5 mRNA, in situ hybridi zation was carried out. In normal rabbits, the accumulation 1. Yamamoto, T. and Bujo, H. (1996) Close encounters with of hybridization signals for LRP5 transcripts, appearing apolipoprotein E receptors. Curr. Opin. Lipidol. 7, 298 302 red, was detected most intensely in parenchymal cells of 2. Yamamoto, T., Davis, C.G., Brown, M.S., Schneider, W.J., the liver (Fig. 4A), and cortical parenchymal cells of the Casey, M.L., Goldstein, J.L., and Russell, D.W. (1984) The human LDL receptor: a cysteine-rich protein with multiple Alu adrenal gland (Fig. 4C), but no significant accumulation was sequences in its mRNA. Cell 39, 27 38 detected in the adrenal medulla (Fig. 4D). These data 3. Ishii, H., Kim, D.H., Fujita, T., Endo, Y., Saeki, S., and suggest that LRP5 may provide cells performing active Yamamoto, T.T. (1998) cDNA cloning of a new low density

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lipoprotein receptor-related protein and mapping of its gene genome. Proc. Natl. Acad. Sci. USA 93, 1320-1324 (LRP3) to bands 19q12.13.2. Genomics 51,132-135 8. Kim, D.H., Iijima, H., Goto, K., Sakai, J., Ishii, H., Kim, H.J., 4. Tomita, Y., Kim, D.H., Magoori, K., Fujino, T., and Yamamoto , Suzuki, H., Kondo, H., Saeki, S., and Yamamoto, T. (1996) T.T. (1998) A novel low density lipoprotein receptor related Human apolipoprotein E receptor 2. A novel lipoprotein receptor protein with type II membrane protein like structure is abundant of the low density lipoprotein receptor family predominantly in heart. J. Biochem. 124, 784-789 expressed in brain. J. Biol. Chem. 271. 8373-8380 5. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular 9. Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., and Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Yamamoto, T. (1992) Rabbit very low density lipoprotein Laboratory, Cold Spring Harbor, NY receptor: a low density lipoprotein receptor-like protein with 6. Sasano, H., Uzuki, M., Sawai, T., Nagura, H., Matsunaga, G., distinct ligand specificity. Proc. Natl. Acad. Sci. USA 89, 9252- Kashimoto, 0., and Harada, N. (1997) Aromatase in human bone 9256 tissue. J. Bone Miner. Res. 12, 1416-1423 10. Hey, P.J., Twells, R.C.J., Phillips, M.S., Nakagawa, Y., Brown, 7. Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Sato, Y., S.D., Kawaguchi, Y., Cox, R., Xie, G., Dugan, V., Hammond, H., Takamori, K-, Tokuda, C., and Saito, 1. (1996) Efficient genera Metzker, M.L., Todd, J.A., and Hess, J.F. (1998) Cloning of a tion of recombinant adenoviruses using adenovirus DNA-ter novel member of the low-density lipoprotein receptor family. minal protein complex and a cosmid bearing the full-length virus Gene 216, 103-111

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