The gene for the human putative apoE receptor is on chromosome 12 in the segment q13–14

GENOMICS

5.65-69

(1989)

The Gene for the Human Putative apoE Receptor Is on Chromosome 12 in the Segment ql3-14 OLA MYKLEBOST,* KRISTINA ARHEDEN,t SISSEL ROGNE,$ AD GEURTS VAN KESSEL,~ NILS h/hNDAHL,t JOACHIM HERZ,~~ KEITH STANLEY,” SVERRE HEIM,t AND FELIX MITELMANt *Biochemistry Department, Institute for Cancer Research, Montebello, N-03 70 Oslo 3, Norway; tDepartment of Clinical Genetics, University Hospital, S-22 1 85 Lund, Sweden; Slnstitute for Animal Science, Agricultural University, N- 1432 .&-NLH, Norway; SDepartment of Cell Biology and Genetics, Erasmus University, NL 3000 DR Rotterdam, The Netherlands; and !European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, Federal Republic of Germany Received

November

14, 1988; revised

2, 1989

are indications that apoE, the probable ligand of LRP,l is involved in immunoregulation and modulation of cell growth and proliferation, in addition to its role in lipid metabolism (Mahley, 1988). The strong homologies between the LRP and the LDL receptor suggest a common origin by gene duplication processes. We have now localized the LRP gene to a chromosome different from that of the LDL receptor gene.

We have previously described the cDNA coding for a new lipoprotein receptor that contains domains closely related to the ligand-binding domain of the LDL receptor. We have now investigated the localization of the gene for this new receptor by hybridization of the cDNA to panels of rodent cells containing subsets of human chromosomes and by in situ hybridization of the cDNA to chromosomes. The gene maps to 12q13-14, a known hot spot for chromosomal rearrangements in human neoplasia. Of particular interest is the frequent involvement of the 12q13-14 segment in clonal abnormalities in lipomas and myxoid liposarcomas, and it is possible that LRP may play a role in the pathogenesis of such tumors. 0 1989 Academic Press,

March

MATERIALS

AND

METHODS

Hybrid Cell Lines The hybrid cell lines were derived from interspecies fusions of rodent and human cells and have been described previously (Geurts van Kessel et al., 1983; van% Veer et al., 1984). The mouse hybrid cell panel was made from mouse myeloid cells (WEHI-TG) and the hamster panel from hamster a3 cells fused with leukocytes from various human donors.

Inc.

INTRODUCTION

The putative apoE receptor, or low-density lipoprotein receptor-related protein (LRP), is a very large protein (MW 500 kDa) with a wide tissue distribution (Herz et al., 1988). The extracellular portion of the protein resembles four repeated copies of the LDL receptor, attached to the cell by a single membranespanning domain. The domains homologous to the ligand-binding domain of the LDL receptor contain highly conserved disulfide bonds and negative charges, and, like the LDL receptor, LRP binds calcium with high affinity. The extracellular portion of the protein also contains repeated domains homologous to the epidermal growth factor (EGF) precursor and to EGF itself. One of these domains is situated in exactly the same position relative to the membrane-spanning segment of LRP as that of the repeat of the EGF precursor which forms the active EGF on proteolytic processing (Scott et al., 1983; Pfeffer and Ullrich, 1985). There

Gene Probe Concatenated purified cDNA fragments spanning 5.6 kb of coding sequence (clones LRP-1, -2, and -4) (Herz et al., 1988) were used as probes for the LRP gene. The fragments were purified after separation on agarose gels by the use of a DEAE membrane (Schleicher & Shuell NA45) and labeled by random primed synthesis after ligation. For hybridization to DNA blots, the probe was labeled to >lOg cpm/pg with [(r-32P]dCTP (3000 Ci/mmol). For in situ hybridization to chromosomes, a specific activity of 3.3 X lo8 cpm/pg was obtained, using [a-3H]dCTP (50 Ci/mmol), [cu-3H]dATP (40 Ci/mmol), and [(u-~H]TTP (103 Ci/mmol). ’ Antibodiesto a cytoplasmictail peptidefrom apoE-binding proteinof the same molecular weight HepG2 liver cells (W. Weber, unpublished). 65

J. Herz,

U. Beisiegel,

LRP identify an in extracts from and K. K. Stanley,

osss-7543/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

66

MYKLEBOST

ET

A 123456

7

6

s 10 9

11

12

13

14

15

AL.

B

16

kb

1

2

3

4

5

6

7

8

9

10

11 *

-

14

--

8.4 6.4

:;

-

4.3

"I( ,>

-

2.3

kb

* -

‘b"

-

u

23.1 9.4 6.6

-

4.4

-

2.3 2.0

FIG. 1. Autoradiograms of DNA blots of the rodent/human hybrid panels hybridized to the human LRP probe. (A) DNA from mouse/ human hybrid cells digested with EcoRI. Samples 1, 4-6, 10-13, and 15 were scored as positive. Lane 16 contains human control DNA. (B) DNA from hamster/human hybrid cells digested with BarnHI. Samples 1-6 were scored as positive. Lanes 9 and 11 contain hamster and human control DNAs, respectively. 25 pg of rodent and 5 pg of human DNA were used for each sample. Lane 10 contains the molecular weight markers. Twenty of the hybrids were karyotyped and used for concordancy analysis (Table 1).

DNA

Blots

TABLE

DNA blots were made from 0.8% agarose gels, and nylon membranes were used as previously described (Rogne et al., 1987). The completion of restriction digests was confirmed by running an aliquot of each digest, incubated with intact X as an internal digestion control, on a checking gel. The blots were hybridized to the probe in 5X SSC, 100 pg/ml salmon sperm DNA, 5% dextran sulfate, and 1% SDS overnight at 65°C and washed down to 0.2X SSC (30 n&f Na+) at 68’C. Autoradiograms were made by exposure of X-ray film (Kodak XAR-5) to the filters for 16-40 h at -70°C with intensifying screens (Agfa MR400). In Situ Hybridization Prometaphase chromosome preparations were made by double synchronization of normal blood lymphocytes from a healthy male donor (Tonne, 1985). Slides with a high mitotic index were aged for 4 weeks, and then in situ hybridization was performed as described in detail previously (Arheden et al., 1988). Eight slides were hybridized at 42°C for 16 h at two different probe concentrations, 0.025 and 0.125 ng/pl. The preparations were coated with Kodak NTBII photographic emulsion in total darkness and stored at 4°C for 10 days. G-banded chromosomes (Cannizzaro and Emanuel, 1984) were analyzed directly in the microscope for the presence of silver grains overlying or in close contact with the chromatids. RESULTS

Hybrid Cell Analysis Autoradiograms from hybridization of the human LRP cDNA probe to filters containing digested DNA

1

Concordances between Detection of the HumanSpecific Bands of the LRP Gene and the Presence of the Different Human Chromosomes in the Hybrid Cell Panels Chromosome/gene concordances

Chromosome 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

Concordance

Discordance

+/+

-/+

10 9 9 I 6 10 9 11 7 9 5 13 9 10 7 6 7 4 5 11 7 9 8

-/5 7 5 4 7 2 5 3 5 3 4 6 6 4 6 5 2 4 6 3 2 4 1

3 4 4 6 7 3 4 2 6 4 8 0 4 3 6 I 6 9 8 2 6 4 5

+/2 0 2 3 0 5 2 4 2 4 3 1* 1 3 1 2 5 3 1 4 5 3 6

Percentage discordance for LRP on chromosome 25 20 30 45 35 40 30 30 40 40 55 5 25 30 35 45 55 60 45 30 55 35 55

Note. The one cell line (WEWI-10) discordant with the LRP gene being on chromosome 12 is marked by an asterisk. Only about 10% of these cells contained chromosome 12. +/+, Positive for chromosome and LRP gene; -/-, neither chromosome nor LRP gene present; +/-, positive for chromosome but not for LRP gene; -/+, negative for chromosome and LRP gene present.

THE

LRP

GENE

IS ON

from the rodent/human hybrid panels are shown in Fig. 1. The results from the mouse/human panel were equivocal, and a hamster/human hybrid panel was used to firmly establish the chromosomal localization. Even under high stringency (0.1X SSC, 68°C) the probe also hybridized to the endogenous rodent gene fragments. The human fragments on the rodent background can be discerned either by deducting bands common to all hybrid lines or by comparing the hybrids with normal human and rodent control samples. Thus, the 14-kb band in the EcoRI digests and the two bands below 2 kb in the BumHI digests are human-specific. The results are summarized in Table 1, where the hybridization pattern of the human-specific bands can be compared with the chromosomes detected in the respective cell lines. The results from 19 of the 20 cell lines that were adequately karyotyped are consistent with the LRP gene being on human chromosome 12. One discordant cell line (WEWI-10, Fig. lA, lane 14) did not hybridize detectably to the LRP probe, although a low proportion of the cells (about 10%) contained

FIG.

2.

G-banded

metaphase

plate

showing

CHROMOSOME

67

12

chromosome 12. Most probably, the hybridization signal from the fraction of these cells containing chromosome 12 is too weak to be detected above the level of background. When less than 10% of the cells contain a certain human chromosome, that cell line is scored as negative for the chromosome. Figure 1B (lane 8) shows that the LRP probe does not hybridize to DNA from a hybrid cell line (van’t Veer et aZ., 1984) containing only the ql4-qter segment of human chromosome 12; thus the LRP gene must reside in the pter-q14 region. In Situ Hybridization A total of 101 metaphases, in which every grain could be localized to a chromosome band, were analyzed. An additional 37 metaphases showing specific hybridization to chromosome 12 were studied with regard only to the grain localization on that chromosome. The number of grains detected in the 101 cells was 161, giving an average of 1.6 grains/metaphase. Fortyfive percent of the metaphases showed specific hybrid-

specific

hybridization

to 12q14

(arrow).

68

MYKLEBOST

ization to chromosome 12 (Fig. 2). Of the 161 grains, 67 (42%) were assigned to chromosome 12 (Fig. 3), and 43 (27%) to the segment 12q13-14. More specifically, 14 grains (9%) were localized proximally in 12q13, 6 (4%) to 12q13.3-14.1, and 23 (14%) to the distal part of 12q14. No other chromosome showed more than a total of 3 grains assigned to a specific band. In the 37 metaphases analyzed only for grains on chromosome 12, a total of 47 grains were scored. Ten (21%) were assigned to the proximal part of 12q13,lO (21%) to 12q13.3-14.1, and 20 (43%) to the distal part of 12q14. These data, combined with those from the 101 metaphases analyzed for hybridization on all chromosomes, give a total of 114 grains localized to chromosome 12. Of these, 24 grains (20%) were localized proximally within 12q13,16 (14%) to 12q13.3-14.1, and 43 (38%) distally in 12q14 (Fig. 4). DISCUSSION

The strong homology between the LRP and the LDL receptor indicates a common origin, perhaps involving duplication of a primordial receptor gene. It seemsunlikely that such complex similarity of structural motif patterns could arise from convergent functional evolution of completely unrelated genes.However, we show that the genes are localized to different chromosomes, as the hybrid panel results are consistent with the LRP probe hybridizing only to the 12pter-q14 region. The reliability of the hybrid panel is indicated by its previous use to successfully localize the INTl gene, localized in the same region, to chromosome 12 (van’t Veer et al., 1984). The in situ hybridizations confirm the chromosomal localization and narrow it down to 12q13-14. The spreading of the grains within the two chromosomal bands could indicate that there are other closely related sequences within this region, but might also simply reflect the limited resolution of the technique. It is possible that there are other genes or pseudogenes ho-

1

2

3

FIG. 3. metaphases

4

5

6

7

8

9

10

11

12

ll*ll,lll 13 14

I 15

16

17

18

19

Chromosomal distribution of 161 grains analyzed for grains on all chromosomes.

20

21

22

in the

x

Y

101

ET

AL.

P q ‘:; 15

l--l12 FIG. mosome

of the 114 grains 4. Distribution 12 in 138 metaphases.

that

hybridized

to chro-

mologous to the LRP gene. However, at the high stringency used, no hybridization to the LDL receptor gene (chromosome 19, cf. Fig. 3) or to any other RNA species in any of the many tissues examined (results not shown) has been detected. The LRP gene is thus, by cytogenetic resolution, localized to a chromosomal region involved in rearrangements in a variety of tumors. In myxoid liposarcomas the specific rearrangement is the translocation t(12;16)(q13;pll) (Turc-Care1 et al., 1986). One-third of the clonal abnormalities in lipomas affect 12q13-14, most often as t(3;12)(q27-2&ql3-14) (Mandahl et al., 1987, 1988). Half of the cytogenetic abnormalities found in uterine leiomyomas are characterized by the reciprocal translocation t(12;14)(q14-15;q23-24) (Heim et al., 1988; Turc-Care1 et al., 1988, Nilbert et al., 1989). Clonal aberrations of 12q13-15, usually translocations but occasionally deletions, are also found in pleomorphic adenomas of the salivary gland (Bullerdiek et al., 1987; Mark et al., 1988). It is possible that some of these 12q breakpoints affect the same gene and that the activation of an oncogenic potential of the rearranged gene could be a common pathogenic mechanism. We and others have previously examined the structures of three other genes localized to this region, the putative oncogenes INTl and GLI and the collagen gene COLBAl. In those cases no structural aberrations could be found when DNA blots from myxoid liposarcomas, lipomas or leiomyomas with cytogenetic aberrations of 12q13-15 were analyzed with the available gene probes (Turc-Care1 et al., 1987; Arheden et al., 1989a,b). Similarly, no aberrations were observed when the LRP probe was used to analyze DNA blots from lipomas with clonal abnormalities of 12q13-15 (results not shown). However, because the LRP cDNA probe covers only part of the 15-kb mRNA, and no flanking regulatory sequences, there may be rearrangements that were not detected. The growth factor-like domains of LRP suggest a role in growth regulation, and its probable importance for lipid metabolism makes it a particularly interesting

THE LRP GENE IS ON CHROMOSOME

candidate gene in lipoma and liposarcoma tumorigenesis. However, other genesinvolved in lipid metabolism have been found localized in clusters (Karathanasis, 1985; Myklebost and Rogne, 1988), and it is not unlikely that genes important for adipose cell function, other than LRP, could be localized in the same region. ACKNOWLEDGMENTS This work was supported by the Norwegian, Swedish, and Dutch (Koningin Wilhelmina Fonds) Cancer Societies, the Swedish Work Environment Fund, and the JAP Foundation for Medical Research. REFERENCES 1. ARHEDEN, K., MANDAHL, N., STROMBECK, B., ISAKSSON, M., AND MITELMAN, F. (1988). Chromosome localization of the human oncogene INTl to 12q13 by in situ hybridization. Cytogenet. Cell Genet.

47: 86-87.

2. ARHEDEN, K., NILBERT, M., HEIM, S., MANDAHL, N., AND MITELMAN, F. (1989a). No amplification or rearrangement of INTl, GLI, or COL2Al in uterine leiomyomas with t(12;14) (ql4-15;q23-24). Cancer Genet. Cytogenet., in press. 3. ARHEDEN, K., MANDAHL, N., HEIM, S., AND MITELMAN, F. (1989b). The INTl oncogene is not rearranged or amplified in lipomas with structural chromosomai abnormalities of 12q1315. Submitted for publication. 4. BULLERDIEK, J., BARTNITZKE, S., WEINBERG, M., CHILLA, R., HAUBRICH, J., AND SCHLOOT, W. (1987). Rearrangements of chromosome region 12q13-15 in pleomorphic adenomas of the salivary gland (PSA). Cytogenet. CeU Genet. 45: 187-190. 5. CANNUZARO, L. A., AND EMANUEL, B. S. (1984). An improved method for G-banded chromosomes after in situ hybridization. Cytogenet.

Cell Genet.

38: 308-309.

6. GEURTS VAN KESSEL, A. H. M., TETTEROO, P. A. T., VON DEM BORNE, A. E. G., HAGEMEIJER, A., AND BOOTSMA, D. (1983). Expression of human myeloid-associated surface antigens in human-mouse myeloid cell hybrids. Proc. N&Z. Acad. Sci. USA 80: 3748-3752. 7. HEIM, S., NILBERT, M., VANNI, R., FLODBRUS, U.-M., MANDAHL, N., LIEDGREN, S., LECCA, II., AND MITELMAN, F. (1988). A specific translocation, t(12;14)(q14-15;q23-24), characterizes a subgroup of uterine leiomyomas. Cancer Genet. Cytogenet. 32: 13-17. 8. HERZ, J., HAMANN, U., ROGNE, S., MYKLEBOST, O., GAUSEPOHL, H., AND STANLEY, K. K. (1988). Surface location and high affinity for calcium of a 500 kDa liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J 7: 4119-4127. 9. KARATHANASIS, S. K. (1985). Apolipoprotein multigene family: Tandem organization of human apolipoprotein AI, CIII, and AIV genes. Proc. Natl. Acad. Sci. USA 82: 6374-6378. 10. MAHLEY, R. T. (1988). Apolipoprotein E: Cholesterol transport protein with expanding role in cell biology. Science 240: 622630.

12

69

11. MANDAHL, N., HEIM, S., JOHANSSON, B., BENNET, K., MERTENS, F., OLSSON, G., ROOSER,B., RYDHOLM, A., WILLBN, H., AND MITELMAN, F. (1987). Lipomas have characteristic structural chromosomal rearrangements of 12q13-q14. Znt. J. Cancer 39: 685-688. 12. MANDAHL, N., HEIM, S., ARHEDEN, K., RYDHOLM, A., WILL~N, H., AND MITELMAN, F. (1988). Three major cytogenetic subgroups can be identified among chromosomally abnormal solitary lipomas. Hum. Genet. 79: 203-208. 13. MARK, J., SANDROS, J., WEDELL, B., STENMAN, G., AND EKEDAHL, C. (1988). Significance of the choice of tissue culture technique on the chromosomal patterns in human mixed salivary gland tumors. Cancer Genet. Cytogenet. 33: 229-244. 14. MYKLEBOST, O., AND ROGNE, S. (1988). A physical map of the apolipoprotein gene cluster on human chromosome 19. Hum. Genet.

78: 244-247.

15. NILBERT, M., HEIM, S., MANDAHL, N., FLODBRUS, U.-M., WILLkN, H., AND MITELMAN, F. (1989). Karyotypic rearrangements in twenty uterine leiomyomas. Cytogenet. Cell Genet., in press. 16. PFEFFER, S., AND ULLRICH, A. (1985). Is the precursor a receptor? Nature (London) 313: 184. 17. ROGNE, S., SKRE’ITING, G., LARSEN, F., MYKLEBOST, O., MEvIIG, B., CARLSON, L. A., HOLMQUIST, L., GJONE, E., AND PRYDZ, H. (1987). The isolation of a cDNA clone for human 1ecithin:cholesterol acyl transferase and its use to analyse the genes in patients with LCAT deficiency and fish eye disease. Biochem. Biophys. Res. Commun. 148: 161-169. 18. R&~NNE,M. (1985). Double synchronization of human lymphocyte cultures: Selection for high resolution banded metaphases in the first and second division. Cytogenet. Cell Genet. 39: 292295.

19. SCOTT, J., URDEA, M. S., QUIROGA, M., SANCHEZ-PESCADOR, R., FONG, N., SELBY, M., RUITER, W. J., AND BELL, G. I. (1983). Structure of a moue submaxillary messenger RNA encoding epidermal growth factor and seven related proteins. Science 221: 236-240. 20. TURC-CAREL, C., LIMON, J., DAL CIN, P., RAO, U., KARAKOUSIS, C., AND SANDBERG, A. A. (1986). Cytogenetic studies of adipose tissue tumors. II. Recurrent reciprocal translocation t(12;16)(q13;pll) in myxoid liposarcomas. Cancer Genet. Cytogenet. 23: 291-299. 21. TURC-CAREL, C., PIETRZAK, E., KAKATI, S., KINNIBURGH, A. J., AND SANDBERG, A. A. (1987). The human int-1 gene is located at chromosome region 12q12-12q13 and is not rearranged in myxoid liposarcoma with t(12;16)(q13;pll). Oncogenc Res. 1: 397-405. 22. TURC-CAREL, C., DAL CIN, P., BOGHOSMN, L., TERK-ZAKARIAN, J., AND SANDBERG, A. A. (1988). Consistent breakpoints in the region 14q22-q24 in uterine leiomyoma. Cancer Genet. Cytogenet. 32: 25-31. 23. VAN’T VEER, L. J., GEURTS VAN KESSEL, A. H. M., VAN HEERIKHUlZEN, H., VAN OOYEN,A., AND NUSSE, R. (1984). Molecular cloning and chromosomal assignment of the human homolog of int-1, a mouse gene implicated in mammary tumorigenesis. Mol.

Cell. Biol.

4: 2532-2534.