Proc. Natl. Acad. Sci. USA Vol. 88, pp. 8636-8640, October 1991 Biochemistry

A second thrombospondin gene in the mouse is similar in organization to thrombospondin 1 but does not respond to serum (DNA sequence/exon/intron structure/protein homol/chromosome 17, band A3)

PAUL BORNSTEIN*t, SREELEKHA DEVARAYALU*, PAUL LI*, CHRISTINE M.

DISTECHEO,

AND PAUL

FRAMSON*

Departments of *Biochemristry and *Pathology, University of Washington, Seattle, WA 98195

Communicated by Russell F. Doolittle, July 11, 1991 (received for review May 15, 1991)

A second, expressed thronbospondin (TSP) ABSTRACT gene, Thbs2, has been identified in the mouse. The exon/intron organization of Thbs2 is hgy conserved in comparison with Thbsl in that exon size and the pattern of interruption of the reading frame by introns are preserved, but there is a marked divergence in coding sequence, pnmarily in the first 7 exons. On the other hand, the DNA and translated amino add sequences of exons coding for the type I, H, and III repeats in the two TSPs are far better conserved. Thbs2 is located on chromosome 17, band A3, whereas Thbsl was found on chromosome 2, band F. In marked contrast to Thbsl , the Thbs2 gene is not induced by serum in NIH 3T3 cells; promoter sequences in the two genes are also very different. It is therefore likely that the two TSPs perform related but distinct functions.

Thrombospondin (TSP) was first identified as a component of platelet a granules (1) but is now recognized as a secreted product ofdiverse cells, including fibroblasts, smooth muscle cells, and endothelial cells (2). Although there is evidence that TSP can serve as an attachment factor for some cells [see Murphy-Ullrich and Hook (3) and Prater et al. (4) for recent discussions], TSP also functions more dynamically in modulating cell-matrix interactions (5). The TSP gene undergoes a rapid response, demonstrated at the level of transcription and mRNA accumulation, to stimulation by mitogens, including serum, platelet-derived growth factor, and basic fibroblast growth factor (6-8), a feature not commonly associated with secreted structural proteins. TSP was thought to be the product of a single gene locus (9-15), but we have recently described a second, homologous cDNA in the mouse that is encoded by a separate gene, as indicated by Southern blot analysis (16). We report here the isolation and partial characterization of this second gene, termed Thbs2, and show that it differs in a fundamental characteristic from Thbsl in its lack of serum responsiveness. The two genes are clearly homologous in structure but show an interesting pattern of differences in coding sequence that predicts similarities as well as differences in the properties of the two proteins.§

restriction maps with Sal I, BamHI, and EcoRI (cloning sites that flank the dispensible sequence in AEMBL3), the three clones were shown to contain overlapping DNA inserts encompassing a total of 22 kilobases (kb) of DNA. The orientation of the genomic clones, relative to the direction of transcription in Thbs2, was ascertained by Southern blot analysis of restriction digests using several probes whose positions in the cDNA sequence were known. DNA Sequence Analysis. Appropriate DNA fragments were subcloned into phagemid vectors and sequenced as doublestranded DNA by the dideoxy chain-termination method, either manually or with an Applied Biosystems model 373A sequencer. In the latter case, dye primer and dye terminator cycle sequencing kits (Applied Biosystems) were used. Sequences were extended using synthetic oligonucleotides as primers. The sequence of the 5' flanking region and first exon was derived from multiple analyses on both strands. DNA and translated amino acid sequences were analyzed using GENEPRO (Riverside Scientific, Seattle) programs. Mapping of the Thbs2 Gene by in Situ Hybridization. The two cDNA clones, pMTSP2-A and pMTSP2-B, were used as probes for mapping. The probes were labeled by nicktranslation to a specific activity of 3.3 x 107 cpm/,ug for pMTSP2-A and 1.8 x 107 cpm/,ug for pMTSP2-B. In situ hybridization to metaphase chromosomes from a male mouse (C57BL/6J) was carried out using a mixture of the two probes, each at 0.02 ug/dul of hybridization mixture, as described (18). The slides were exposed for 14-28 days, and chromosomes were identified by Q-banding. Analysis of Serum Responsiveness of Thbsl and Thbs2 Genes. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum. Cells were made quiescent by culture in medium containing 0.4% serum for 24 hr. At different times after a change of medium containing 15% fetal calf serum, RNA was extracted and concentrations of mRNA for Thbsl and Thbs2 were determined by an RNase protection assay (19, 20). Specific antisense riboprobes were generated from a 272-base-pair (bp) mouse genomic fragment that protects 181 bp of exon 5 in Thbsl mRNA (15) and from a 267-bp mouse genomic fragment that protects 142 bp of exon 5 in Thbs2 mRNA.

MATERIALS AND METHODS Isolation and Characterization of Thbs2 Genomic Clones. Mouse genomic DNA was partially digested with Mbo I and cloned into the BamHI site of AEMBL3 phage to produce a genomic library. Plaques (6 x 105) were screened separately with random primer-labeled cDNA probes, pMTSP2-A (nucleotides 1-479) and pMTSP2-B (nucleotides 835-1348), of Thbs2 cDNA (16); three clones that hybridized with both probes were purified. Phage DNA was prepared by liquid lysis and purified on a glycerol step gradient (17). From

RESULTS Exon/Intron Organization of the Thbs2 Gene. A map of approximately the first two-thirds of the mouse Thbs2 gene is shown in Fig. 1. The orientation of the gene with respect to the direction of transcription was established by Southern blot analysis of restriction digests using cDNA probes derived approximately from (i) exons 1-3, (ii) exons 4-7, and Abbreviations: TSP, thrombospondin; MTSP1, mouse TSP1; MTSP2, mouse TSP2; HTSP, human TSP. tTo whom reprint requests should be addressed. §The sequences reported in this paper have been deposited in the GenBank data base (accession no. M67455).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 88 (1991)

E

121

B

- 9240

1116

0o

-10

1 1000 bp

97

6- 700 5

-9U

-1900

3

B 141

-1500

E 85

4

B 171

-1850

8f 249

i

174k

177

I

-750

10

8637

197

-M -1800 -

S

1219

128 153

1112

-300

76

13 -850

FIG. 1. Map of the Thbs2 gene through exon 13. The start of transcription is indicated by an angled arrow. Exons are indicated by rectangles and introns and 5' flanking sequence by lines. Untranslated exons are shown as open rectangles and coding sequence as filled rectangles. Size (in bp) is shown above exons and below introns and the 5' flanking sequence. Most intron sizes are estimates based on partial sequence and restriction maps, but introns 8 and 11 were sequenced completely. All BamHI (B), EcoRI (E), and Sal I (S) sites in the sequence are indicated.

(iii) exons 13 and 14. The strategy that proved most effective in locating exonic sequences quickly was to identify rare restriction sites within the previously established cDNA sequence for this part of the gene (16) and to subclone genomic fragments utilizing one of these and adjacent genomic restriction sites. As an example, an Apa I site in exon 8 was used to produce two Apa I/BamHI fragments extending upstream and downstream from exon 8 (Fig. 1). DNA sequence analysis, initiated in both directions from the Apa I site, provided the sequence of exon 8 together with that of the exon/intron boundaries and also provided the sequence of exon 9, since the length of intron 8 is short (249 bp). A striking feature of the structure of Thbs2 is that the size and relation to the reading frame of coding exons are highly conserved when compared with either Thbs2 or THBSI (Table 1). Starting with exon 9 and progressing in a 3' direction, the exon sizes are identical; for exons 3, 5, 7, and 8, the differences reflect the addition or subtraction of single codons; for exons 4 and 6, the differences are three and six codons, respectively. Thus, the phase class-i.e., the position in the codon in which introns interrupt coding sequences (21)-is the same in the two TSP genes (Table 1). This conservation would be expected to facilitate exon shuffling between the two genes (21). In every instance, sequences at exon/intron junctions corresponded to splice consensus rules (ref. 22; data not shown). As might be expected, intron size is less conserved between Thbs2 and Thbsl, although it is well conserved between Thbsl and THBSJ (Table 1). It is interesting to note that introns 1-5 in Thbs2 are much larger than most of the corresponding introns in Thbsl, but starting with intron 6 the Table 1. Exon/intron organization in TSP genes Exon size, bp Exon or intron no. Thbs2 Thbsl* THBSOt 1 121 183 150 2 95 149 95 3 557 560 560 4 85 76 76 5 197 200 200 6 141 123 123 7 97 94 94 8 174 171 174 9 177 177 177 10 174 174 174 11 128 ND 128 12 153 ND 153 13 219 ND 219 ND, not determined. *Data from ref. 15. tHuman thrombospondin (HTSP) 1; data from refs. 10, 11,

introns in the TSP1 and TSP2 genes are more similar in size (Table 1). This greater similarity in intron size correlates with a higher degree of identity in DNA sequence in adjacent exons (see below). The 13 exons included in this study correspond to nucleotides 1-2369 in the previously established partial cDNA sequence of Thbs2 (16). A comparison of the DNA and translated amino acid sequences of these exons in the TSP1 and TSP2 genes is presented in Table 2. Since the sequence of Thbsl is known only through exon 10, comparison of exons 11-13 was performed with the corresponding sequences in THBSJ. It can be seen that the degree of sequence identity is relatively low in exons 3-7 (exon 1 is noncoding and exon 2 is partially so) but increases substantially starting with exon 8. Since comparison of exons 11-13 between the TSP1 and TSP2 genes also crosses species lines, one would expect that the degree of identity would be even higher if the sequences in Thbsl were available for comparison. Sequence conservation is low in exon 3, which, based on its size (185 amino acids) and proximity to the NH2 terminus, is likely to contain the heparin-binding domain of TSP in Thbsl (23, 24). The sequences of exons 6 and 7, which together contribute the 55-amino acid sequence that is homologous to the NH2terminal propeptide domain of al(I) procollagen, are not highly conserved, but the procollagen homology remains intact. However, type I, II, and III repeats in exons 8-10, 11-13, and 14-17 are highly conserved in the TSP1 and TSP2 genes (Table 2; ref. 16). Chromosomal Location of Thbs2. The Thbs2 gene was mapped on mouse chromosomes by in situ hybridization. Forty-eight metaphase cells were examined. Of 81 sites of Phase class

Thbs2 -1 1-0 0-1 1-0 0-0 0-1 1-1 1-1 1-1 1-0 0-0 0-0

and 13.

Intron size,

Thbsl*

THBSJt

-1 1-0 0-1 1-0 0-0 0-1 1-1 1-1 1-1 ND ND ND

-1 1-0 0-1 1-0 0-0 0-1 1-1 1-1 1-1 1-0 0-0 0-0

Thbs2 -2400

'1100 "1900 '1800 "1500 -700

'1850 249 -750 -300 76 -850 ND

bp

Thbsl*

THBSJt

527 292 807 244 94 755 -1500 498 280 ND ND ND ND

600 268 >528 319 110 >308 >453 486 2% 247 106 430 468

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Proc. Natl. Acad. Sci. USA 88 (1991)

Table 2. Comparison of cDNA and translated amino acid sequences in Thbs2 with those in Thbsl or THBSI % identity Domain or Exon no. repeat DNA Protein 1 49 2 42 3 49 31 Heparin-binding 4 52 43 5 47 30 6 41 34 Collagen 7 56 38 Collagen I 64 62 8 I 74 69 9 10 I 70 68 II 11 64* 69* 12 II 62* 56* II 13 78* 73* Data for Thbsl and THBSJ are from refs. 9, 12, 13, and 15. *Comparison with THBSL.

A

17

FIG. 2. Distribution of grains, detected by in situ hybridization and autoradiography, as shown on a diagram of mouse chromosome 17. The Thbs2 gene is located on band A3.

hybridization, 23 (28% of the sites) were located on the proximal portion of chromosome 17 (Fig. 2). The largest number of grains was on band A3 of chromosome 17, with some grains on bands A2 and B. There was no significant hybridization to other mouse chromosomes. We conclude that the Thbs2 gene is located on chromosome 17, band A3, or at the A3/B boundary. In contrast, Thbsl is located on chromosome 2 (14). Sequence of the Promoter and First Exon in Thbs2. The sequence of 1116 bp upstream from the start of transcription and of 121 bp in the first, untranslated exon is shown in Fig. 3. A typical TATAAA motif is present 31 bp upstream from the transcription start site. The sequences CGAATT at -123 or CAAAT at -143 might serve as modified CCAAT boxes. No matches were found for a consensus SP1 binding site (GGGCGG) or an AP1 site [TGA(G/C)TC(T/A)]. The first exon contains an upstream ATG followed by an in-frame stop codon (Fig. 3). The translation start site in Thbs2, as in Thbsl, is located in exon 2. A striking finding is the complete lack of similarity in sequence between the 5' flanking sequence in Thbs2 and that in Thbsl (ref. 15 and unpublished data). Thus, when various parameters were used to align 1116 bp upstream of the start of transcription in both genes, the longest stretch of uninterrupted identity was 6 bp (data not shown). Serum Responsiveness of the Thbsl and Thbs2 Genes. The TSP gene has previously been shown to respond rapidly to platelet-derived growth factor and serum, as determined by an increase in protein (25, 26) and mRNA (6, 7). We sought to compare the response of Thbsl and Thbs2 to serum in NIH 3T3 cells after a period of quiescence. The results are shown in Fig. 4. The band identified as MTSP1 in Fig. 4 represents protection of 181 bp in exon 5 of Thbsl . As can be seen, cells in culture in 10% serum contain relatively low levels of -1116 -1040 - 960 - 880 - 800 -

720 640 560 480 400

320 240 160 80 1 80

GGATCC TAATTTATCT TGAGTGTCGT TGCATCACCG CCTGGCACTG CTTGATAACC TGGGCTTTTT TTCTCCTGAG CTATAATAAC CAGTATCACA CCAGCCAATC TTGTCTGACT TTGGACTGGG AACGTGCCTC

AACACAATGG TTCCTTTTCT CTATGAAGAA CCTGACCAAG GCTTTAACCC ACGAGGACTG CAGGGAGCCA AAGCGGGTCA TTCCCGGCAG CTGGGTAATT TTGCTGGTTT CCTCCCCGGG TAACCAGGTA CGCATGTGTC

CAGGGTCCTT TGAAGTAAGC TGAAGCCCAA TCTTTCCTGT CCATGGACTT CCTCTCACTG GGGTCACAGA GAGATACAAT TGTGGTAAAC CGAAACACAT TATTTATAGC CCCCTGTCTG CTCTGAGCAA TGATTGACAG

mRNA for Thbsl (lanes 1 and 2). This level is decreased even further after 24 hr of serum deprivation (lanes 3 and 4). Within an hour after addition of 15% serum, mRNA levels increase 50- to 100-fold (lanes 5 and 6; P.F. and P.B., unpublished data) and this level is maintained for at least 3 hr (lanes 7 and 8). The higher molecular weight, protected band in lanes 5 and 6 is predicted by unspliced nuclear RNA. By 6 hr, mRNA levels have declined appreciably (lanes 9 and 10) and, by 24 hr, levels have returned to those seen in chronic serum stimulation (lanes 11 and 12, compare with lanes 1 and 2). The probe for Thbs2 protects a band of 142 bp in exon 5, a region in which cross-hybridization with Thbsl mRNA is predicted by sequence to be minimal (Table 2). In contrast to Thbsl, mRNA levels for Thbs2 do not change significantly either during quiescence or after serum stimulation (Fig. 4, band MTSP2). Similar findings were obtained in 3T3L1 and BALB/c-3T3 cells, although in the latter cell line the reduction in TSP1 mRNA with serum deprivation was not observed (data not shown). We therefore conclude that, in contrast to Thbsl, Thbs2 is not a serum-responsive gene.

DISCUSSION A second TSP cDNA was recently isolated and characterized in the mouse and was shown to be the product of a separate (16). Based on a comparison of cDNA sequences of Thbsl and Thbs2, we concluded that the two proteins were likely to possess overlapping and distinct properties. In this communication we describe the isolation, partial characterization, and chromosomal location of the Thbs2 gene. Despite very substantial differences in coding sequences, particularly in exons 3-7 (Table 2), the exon/intron structures of Thbsl and Thbs2 are highly conserved. In particular, the interruption of the reading frame by introns occurs in the same position in the codon in the two genes (Table 1). Thus, the potential exists for the exchange of exons between the two

gene

CAGTTAAGTC CCTGACTTAT GAGGAGGTTG TGACTGCTTT AGGTCTGGCC CAGGATTCCC GAATTGCAGG GGGTGGGCTC TTACTGTCTG TTAAAGCATA ATTTTATTTC GAAAGCCTGG ATGGCGAGAC GCCTCCTGGG

TCCCACCTAG AACAGAAAGT GGCTAAGTAC TCTCTITTT TTATTATCAG GTGTCAACCA GATAAATGTC CAGAAAGCTT TGGGAGATGC CACAAATCTA CCATCTTCTT TGGGCTCTGG CCTTGTGTGA TGAGCCCCAT CCCATCCAGC TACCATGCAC CTCTGATTCT CACTGAGTTT CTAAGCTGCC CCCACCCTAT ACACTTGGCA AAGTTGTCCC AACAAGACTG GGGTGAGGAC TCTCGACTGA GCAGATGAAG TGAGTTCAAA TTAAAATCTT TCTTTTGTGG TAAATTCATT AAAAAAAATT AATTCTTTGA TCCTGTAGTT TTGACTGTTA CTCACTTGTT TTAGAAAGGC GAATTGGCAT TTTTTTTTCT CCTCAGCAAA ATGGGGAGTC CTGAGAGAAG ACAGGAAAAG TGAGTCCTGG CCTGCCCCTC GTGGACCTGT AGGAGGGGCG TTAGCTCTGA GGGGCTGGAT CAGAGGACGA ATTATGACAC TCCTTCGAAA GAATGAATTA AT GAAAGGGC AGC CGGTCACTGC CGGGAGGCAG

r-m

AGAGAGCCAG TCCGATGTCT GCAGCCTCCC TGGCCAGGCC TCTCCTCTCC TGCCGCAGCT AGTCCCCCTC AGGACAGACA GAGTACTGGC GTCGGTCACC ATTCACTTGC AAACACACCA G

FIG. 3. DNA sequence of the 5' flanking region and first exon of Thbs2. The sequence extends from a BamHI site at -1116 to nucleotide 121 (nucleotide + 1 represents the first transcribed base). The TATAAA motif is boxed. The sequences of an upstream ATG and an in-frame stop codon in exon 1 are underlined.

Proc. Natl. Acad. Sci. USA 88 (1991)

Biochemisfty: Bomstein et al. 0/ serum

hours after serum

-

2014

-

MTSP1

F

MTSP2

s-

76S

9

3 4 5 6 7 8 9 10 11 12

P2 Pl

FIG. 4. Autoradiogram of a RNase protection assay for TSP niRNA. Total RNA (6,ug, extracted from duplicate dishes of NIH 3T3 cells) was hybridized simultaneously with 32P-labeled antisense Thbsl and Thbs2 genomic probes. After RNase digestion, ethanolprecipitable material was analyzed by electrophoresis in a 6% acrylanide denaturing gel. Lanes 1 and 2, cells grown in 10%o serum; lanes 3 and 4, cells made quiescent for 24 hr in 0.4% serum; lanes 5-12, cells stimulated for various times with 15% serum. Lanes 5 and 6, 1 hr; lanes 7 and 8, 3 hr; lanes 9 and 10, 6 hr; lanes 11 and 12, 24 hr; lane S, Msp I digest of pBR322 as molecular weight standard (in bp); lane P2, mouse TSP2 (MTSP2) riboprobe; lane P1, mouse TSP1 (MTSP1) riboprobe. Arrows indicate the bands specifically protected by these riboprobes.

during evolution (21), although there is no evidence that this event has in fact occurred. We currently do not have an explanation for the tendency of introns in the 5' region of the gene-i.e., introns 1-5-to differ markedly in size between Thbsl and Thbs2, whereas better conservation in size occurs downstream-i.e., introns 6-12 (Table 1). This increased conservation in intron size correlates with an increase in similarity in coding sequence (Table 2). Of equal interest is that the introns in Thbsl and THBS1 are surprisingly similar in size, although the sequences that are available for comparison do not show a high degree of sequence

genes

similarity.

The rather substantial difference in the translated amino acid sequence of the NH2-terminal domains of MTSP1 and MTSP2 raises the possibility that MTSP2 may not be a heparin-binding protein or may bind less well to heparin compared with MTSP1. The heparin-binding domain of TSP1, which comprises the NH2-terminal globular region of the protein (23, 24), is thought to be responsible for binding to heparan sulfate proteoglycans and for mediating some of the functions of TSP (27, 28). On the other hand, other cell surface receptors for TSP probably exist (4, 29-31) and cooperative interactions between receptor systems may occur (32). Thus, the net effect of TSP on cellular function may reflect not only receptor distribution but also the relative extracellular concentrations of the two TSPs. It is also not excluded that heterotrimeric TSP molecules exist-i.e., molecules containing mixtures of TSP1 and TSP2 chains-since the cysteines that are thought to form the interchain disulfide bonds are conserved. However, it is unlikely that TSP is an obligatory heterotrimer since significant differences in the distribution of the two mRNAs can be shown in tissues of the mouse (16). A more practical consequence of the existence of the two protein chains is that the interpretation of studies of immunolocation (33, 34) and of functional neutralization (35) could be complicated by the extent to which antibodies interact with each of the TSPs. The Thbs2 gene maps near the t complex in a region just proximal to the H2 major histocompatibility complex on mouse chromosome 17. The human THBS2 gene maps to the terminal region of the long arm of chromosome 6 (T. LaBell, C.M.D., and P. H. Byers, unpublished data); this finding

8639

confirms the existence of conservation between the proximal region of mouse chromosome 17 and the terminal region of human chromosome 6, as defined by the location of the Tcpl (t-complex protein 1) gene (36-38). A striking difference exists in the response of the Thbsl and Thbs2 genes to serum in NIH 3T3 cells. Whereas the Thbsl gene is rapidly induced by a factor of 50-100 in quiescent cells exposed to serum, no significant change in mRNA levels for Thbs2 was observed (Fig. 4). Although we cannot exclude a rapid compensatory increase in degradation of Thbs2 mRNA, it seems most likely that the Thbs2 gene lacks the cis-acting elements that mediate the serum response in Thbsl. The sequence of the first 1116 nucleotides of the 5' flanking sequence in MTSP2 (Fig. 3) shows little or no similarity to the analogous region in Thbsl and this difference extends further upstream. In particular, a putative serum-response element, located at -1290 in THBSI (10,11), is conserved in Thbsl but is missing in Thbs2 (data not shown). The marked difference in regulation of expression of the two TSP genes underscores the likelihood that the products of these homologous genes will be found to have related but distinct properties. Just prior to submission of this manuscript for publication, a report appeared describing the cDNA sequence of chicken TSP (39). The cDNA was considered to be the chicken homologue of human and mouse TSP1, but it is apparent from a comparison with the sequence of the mouse TSP2 cDNA (16) that the chicken sequence is, in fact, that of TSP2. A preliminary analysis indicates that the degree of amino acid sequence identity between mouse and chicken TSP2 is almost as great as that between human and mouse TSP1. This finding supports our suggestion that the sequence differences between TSP1 and TSP2 will be found to be functionally

significant. We thank Richard Palmiter for generously providing the mouse genomic library and David Adler for his help with the mapping studies. We also thank Helene Sage for a critical review and Kathleen Doehring for preparation of the manuscript. Oligonucleotide synthesis and automated DNA sequencing were carried out by the University of Washington Molecular Pharmacology Facility. This work was supported by National Institutes of Health Grants HL-18645, DE-08229, and AG-01751 and by a grant from the March of Dimes (1-1019). 1. Baenziger, N. L., Brodie, G. N. & Majerus, P. W. (1972) J. Biol. Chem. 247, 2723-2731. 2. Majack, R. A. & Bornstein, P. (1987) in Cell Membranes, eds. Elson, E., Frazier, W. & Glaser, L. (Plenum, New York), Vol. 3, pp. 55-77. 3. Murphy-Ulirich, J. E. & Hook, M. (1989) J. Cell Biol. 109, 1309-1319. 4. Prater, C. A., Plotkin, J., Jaye, D. & Frazier, W. A. (1991) J. Cell Biol. 112, 1031-1040. 5. Sage, H. & Bornstein, P. (1991) J. Biol. Chem. 266, 1483114834. 6. Kobayashi, S., Eden-McCutchan, F., Framson, P. & Bornstein, P. (1986) Biochemistry 25, 8418-8425. 7. Majack, R. A., Milbrandt, J. & Dixit, V. M. (1987) J. Biol. Chem. 262, 8821-8825. 8. Donoviel, D. B., Amacher, S. L., Judge, K. W. & Bornstein, P. (1990) J. Cell. Physiol. 145, 16-23. 9. Lawler, J. & Hynes, R. 0. (1986) J. Cell Biol. 103, 1635-1648. 10. Donoviel, D. B., Framson, P., Eldridge, C. F., Cooke, M., Kobayashi, S. & Bornstein, P. (1988) J. Biol. Chem. 263, 18590-18593. 11. Laherty, C. D., Gierman, T. M. & Dixit, V. M. (1989) J. Biol. Chem. 264, 11222-11227. 12. Hennessy, S. W., Frazier, B. A., Kim, D. D., Deckwerth, T. L., Baumgartel, D. M., Rotwein, P. & Frazier, W. A. (1989) J. Cell Biol. 108, 729-736. 13. Wolf, F. W., Eddy, R. L., Shows, T. B. & Dixit, V. M. (1990) Genomics 6, 685-691.

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