The Murine Urokinase-type Plasminogen Activator Receptor Gene*

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Apr 25, 1994 - The murine urokinase-type plasminogen activator re- ceptor (uPAR) gene has been isolated and its complete nucleotide sequence established ...
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biolom, Inc.

Vol. 269, No. 42, Issue of October 21, pp. 25992-25998, 1994 Printed in U.S.A.

The Murine Urokinase-type Plasminogen Activator Receptor Gene* (Received for publication, April 25, 1994, and in revised form, July 18, 1994)

Theodore T. SuhSO, Claus Nerlofl, Keld Danan, and Jay L. DegenSII From the $Division of Basic Science Research, Children’s Hospital Research Foundation, Cincinnati, Ohio 45229 and the llFinsen Laboratory, Righospitalet, Strandboulevarden 49, 2100 Copenhagen0, Denmark

The murine urokinase-type plasminogen activator re- esses and isknown to actat the cell surface througha specific ceptor (uPAR) gene has been isolated and its complete saturable cell surface receptor. However, despite the associanucleotide sequence established. The gene is organized tion of uPA with tissue remodeling events (lo), aswell as the into seven exons comprising 9.5%of the 13,207-base pair intricate regulation of uPA activity at the level of gene expresregion that spans the interval betweenthe transcription sion (101, proenzyme activation (11, 12), serpin inhibition (10initiation andpolyadenylation sites. The region up- 121, and receptor-mediated localization on the cell surface (10stream of the transcription initiation site lacks TATA- or 12), uPA-deficient transgenic mice appear to develop normally CCAAT-like elements but is flanked by a G+C-rich region, which contains a number of potential regulatory and exhibit remarkably limited phenotypic abnormalities in elements including Spl and AP1 binding motifs. The the absence of other challengingfactors (13). A detailed understanding of the role of the urokinase-type close association of both Spl and AP1 sites within the proximal promoter regionis consistent with the obser- plasminogen activator receptor (uPAR) will be fundamental to vation thatthe murine uPAR gene is inducible by phor- resolving the precise physiological and pathologicalroles of bo1 esters. The majorfunctional domainsof the encoded uPA-mediated proteolysis. uPAR specifically binds the active protein, including the signal peptide, three cysteine- two-chain formof uPA (141,as well as any of the inactive forms rich internal repeats, and the glycolipid anchor attach- of uPA: pro-uPA (14) and uPA-plasminogen activator inhibitor ment motif, are encoded by separate exons. Based on the complexes (15). The significance of uPAR in the plasminogen organization of the murine UPAR gene andthe distribu- activation system is that it provides a means of focusing plastion of protein domains within the exons in the Ly-6 min formation at the cell surface (16-18), as well as a mechafamily of genes, it appears that the uPAR gene evolved nism for internalizing uPA-inhibitor complexes (3, 19). secondarily to two internal duplicationevents within a uPAR is a highlyglycosylated,55-60-kDa integral memLy-6-like ancestralgene. The cloned and sequenced mu- brane protein (20) linked to the plasma membraneby a glycorine uPAR gene will be a valuable tool in understanding the regulation and biological roles of uPARin that it will sylphosphatidylinositol (GPI) anchor (21). The pattern of dipermit detailed studies of gene expression and uPAR- sulfide cross-links in the amino-terminal domain place uPAR dependent processes in uitro, as well as the generation within a superfamily of GPI-anchored proteins (22) whose Ly-6 antigens (231, the membrane of both gain-of-functionand loss-of-function mutants in members include the murine inhibitor of reactive lysis (MIRL) orCD59 (241,the squid brain transgenic mice. glycoprotein Sgp-2 (25), a lymphotropic tumor virus protein designated HVS-15 (26), and a family of snake neurotoxins represented by erabutoxin C (27). I t has been shown that the Plasminogen activation focused at the cell surface has been proposed to be critical for both productive extracellular prote- genes encoding MIRUCD59 and Ly-6 share a similar organiolysis and efficient cellular penetration of physical barriers. zation (28-30). Unlike the other superfamily members,uPAR The utility of plasminogen activation is highlightedby the di- contains the characteristiccysteine-rich domain in triplicate. We report the cloning and characterization of the murine verse biological contexts in which it is employed, including uPAR gene. This study provides the first information on the fibrin clot lysis (l),macrophage migration (2, 31, trophoblast implantation (4,5), angiogenesis (61, and tumorcell invasion (7, structural details and organization of the uPAR gene in any 8) and metastasis (9).Urokinase-type plasminogen activator species (see “Note Added in Proof”). These data allow for a (UPA)’has beenspecifically associated with many of these proc- comparison of the uPAR gene with other members of the Ly-6 gene superfamily and suggest a probable mechanism by which the unique repeated proteinmotif evolved. The availability of * This work was supported in part by National Grant-in-aid92-1103 from the American Heart Association (with funds contributed in part by the entire gene and over 10 kilobases of both upstream and the American Heart Association, Ohio affiliate) andby funds from the downstream flanking sequences provides a means by which to Danish Cancer Society(to K. D.). This studywas done during the tenure study the regulatory elements of this gene related to tissue of Established Investigatorship 93002570 (to J. L. D.) from the Amerispecificity (12) and known modulators, such as phorbol esters can Heart Association. The costs of publication of this article were (ll),basic fibroblast growth factor (311, and epidermal growth defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordancewith 18 factor (11). In addition, the gene sequence now makes it posprocesses sible t o dissect the role of the protein domains in the U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to of focal extracellular proteolysis and signal transduction. Furthe G‘enBankm jEMBL Data Bank with accession number(s1 U12235. thermore, the cloned and sequenced murine uPAR gene now 5 Supported by a fellowship from the University of Cincinnati Medimakes itpossible to address the regulation and biological roles cal Science Scholars Program. )I To whom correspondence should be addressed: Children’s Hospital of uPAR using gene targeting and site-directed mutagenesis in Research Foundation, TCHRF2025,3333 Burnet Ave., Cincinnati, OH transgenic mice. 45229-3039. Tel.: 513-559-4679; Fax: 513-559-4317. The abbreviations usedare: uPA, urokinase-type plasminogen actiEXPERIMENTALPROCEDURES vator; uPAFt, urokinase-typeplasminogen activator receptor; GPI, glyIdentification of Recombinant Bacteriophage Containing Murine cosylphosphatidylinositol; bp, base pairb); MIRL, membrane inhibitor Genomic DNAs-A bacteriophage ADASHII genomic DNA library preof reactive lysis; TGF, transforming growth factor.

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Urokinase Receptor Gene r N k l

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FIG.1. Organization of the murine uPAR gene. A, partial map of the restriction endonuclease cleavage sites. The filled areas indicate the location of exons, whilethe open areas indicate the 5'- and 3"noncoding regions, as well as theintrons. The hatched area in exon 4 indicates the region that is included in the mRNA alternative splice variant encoding uPAFt2 (33). B , relative alignment of the DNA inserts from four recombinant bacteriophage carrying portions of the murine u P &gene. C , DNAfragments subcloned into the plasmid vector pUC18that were used for restriction mapping and sequencing of the gene and its surrounding noncoding regions.D , DNA sequencing strategy, illustrated with arrows; the complementary circle-tailedarrows indicate portions sequenced onthe mRNA-like strand, and bar-tailed arrows indicate regions sequenced on strand. pared using DNA from 129/SvJ strain mice was screened by in situ plaque hybridization (32) with various murine uPAR cDNA probes. A 1250-bp PstI fragment of the murine UPAR cDNA (33) that codes for amino acids 24-304 (mature protein numbering system) was used as an initial probe to identify recombinant phage carrying portions of the murine uPAR gene. Subsequently, a 170-bp PstI fragment (containing 5'-flanking sequence, exon 1, and 17 bp of exon 2) and a 150-bp AuaU PuuII fragment (containing most of exon 5 and partof exon 6 ) were used to obtain phage with overlapping portions of the murine uPAR gene. These fragments were independently labeled with [ C E - ~ ~ P I ~by CT the P random primer extension method (34) to a specific activity of 2-5 x 10' c p d p g and used in hybridization mixtures at 1 x lo6 cpdml. Hybridization conditions were at 65 "C in solutions consisting of 6 x SSC (1x SSC = 1.5 m~ sodium citrate, pH 7.0, 0.15 M NaCl), 0.04% Ficoll 400, 0.04%bovine serum albumin, 0.04%poly(vinylpyrrolidone), 1 mM EDTA, and 0.5% SDS. The filters (BA85 nitrocellulose, Schleicher & Schuell) were washed at the same temperature in 1 x SSC, 0.5% SDS and exposed to Kodak X-Omat XAR-5 film overnight at -70 "C with intensifling screens. DNA Sequence Analysis-The methods for constructing and isolating plasmid subclones, as well as mapping restriction endonuclease cleavage sites, have been described previously(35). Protocols forthe preparation of single- and double-stranded DNA templates were adapted from those suggested for the sequencing system using the T7DNA polymerase, SequenaseTM version 2.0 (U. S. Biochemical Corp.)(36,37). The DNA sequence was determined by the Sanger dideoxynucleotide chain termination method (38). Single-stranded M13 DNA templates were made as follows. Briefly, 1.2 ml of M13 phage cultures were harvested and precipitated with 0.25 volumes of 20% polyethyleneglycol 8000, 2.5 M NaCl. The phage were resuspended in 0.2 ml of TE buffer, then extracted twice with equal volumes of phenol, 20% 0.5M Tris-HC1, pH 7.5, and chloroform.A final chloroform extraction was performed before the phage DNA was precipitated by 0.25 volumesof 7.5 M ammonium acetate and 2.5 volumes of ethanol. The single-stranded DNA was obtained after centrifugation, washed with 70% ethanol, and redissolved in 10-20 pl of water, 7 pl of which were used for sequencing. Double-stranded DNA templates were prepared by alkaline denaturation. Miniprep or CsC1-banded plasmid DNAs (2-3 pg) were denatured in 0.2 volumes of 1 M NaOH and incubated at 65 "C for 10 min. The mixtures were then neutralized by adding 0.15 volumes of 2 M sodium acetate, pH 5.2, and the DNA was precipitated with 2.5 volumes of ethanol. After the DNA pellet was washed with 70% ethanol, the DNA was resuspended in 7 pl of water and used for sequencing. ZdentificationofDunscrzption Znitiation Site-The 5' terminus of the murine uPAR mRNA was defined by oligonucleotide primer extension analysis as previously published (39). Two synthetic oligonucleotide primers were used. Exon 1 primer: S'-TGGGACACAGGTAGTCGCCAGCAACAGC-3' Exon 2 primer: S'-GGTTACTCTCACACTGCATGCACTGCAGGC-3'

Briefly, these primers were end-labeled with [y-32PlATP and T4 polynucleotide kinase and were purified on a denaturing polyacrylamide gel. A labeled primer was added to 30-pl hybridization mixtures containing either 15pg of control (yeast tRNA) or 15 pg of poly(AF RNA isolated from J774A. 1 mouse monocyte-macrophage cells (American Type Culture Collection)treated with 160 n~ phorbol 12-myristate 13acetate for 8.5 h. Annealed primer was extended with avian myeloblastosis reverse transcriptase (Life Sciences Inc.) in 20-9 reaction mixtures. Primer extension products were analyzed by electrophoresis on 8%sequencing gels and autoradiography. RESULTSANDDISCUSSION

Isolationand Nucleotide Sequence of theMurine uPAR Gene-A 129/SvJ murine genomic DNA library constructed using the ADASHII cloning vector was screened for clones containing the murine uPAR gene by in situ plaque hybridization using murine uPAR cDNA probes. Four overlapping bacteriophage isolates spanning the entire uPAR protein-coding region and approximately 10 kilobases of upstream flanking sequence and 18 kilobases of downstream sequence were characterized (Fig. 1,A and B ) . The overall organization, placement of restriction endonuclease cleavage sites, and strategy for defining the nucleotide sequence of the murine uPAR gene are presented in Fig. 1.The nucleotide sequence, including 876 bp of 5"flanking sequence and 1936 bp of 3"flanking sequence, is shown in Fig. 2. Of the 16,019 nucleotides sequenced, 50.5% were determined on both strands and 74.4% were established at least twice (Fig. 1 D ) . A comparison of the genomic sequence and the previously reported mouse uPAR cDNAsequence (33) established the general organization of the gene. The size of the murine uPAR gene from the siteof transcription initiation (see below) to the polyadenylation site (33) is 13,207 basepairs. The gene is organized into seven exonsseparated by six intervening sequences (Fig. 2 and Table I). For the GPI-anchored splice variant, muPAR1, the exons comprise9.5% of the gene sequence. The splicejunctions uniformly obey the GT-AG rule and generally follow the consensus splice junction sequences compiled by Mount (40) (Table 11). The Ly-61uPAR Gene Family-Based on the similarity in protein sequence and domain architecture, uPAR has been proposed to be a member of the Ly-6 gene superfamily, among which are the genes encoding the murine Ly-6 antigens (28), MIRL or CD59 (29), and a group of snake neurotoxins represented by erabutoxin C (27). These proteins each contain a signal peptide, one or more cysteine-rich domains that can be

25994

Urokinase Receptor Gene

TCAGGGCTTT TGCGATGACTCTTTCCTCTC CCTCAAAGGC TTTCTGTAGG AATCCACTAG -817 ACTTCTTGCA CACCTTTCCTGGCTGCCCTC CGCATCACCC TCCCAAAGCA ATTAAGCcAT -757 TTACCTCTCT CCCTACAGTTTGTTCCTCTT TCAACTTGTT TTTCCCCTGG TTTCCGACTG -697 ATGGGAGAGA TTAGATAACT AAGTATCCTC CCCATCGTAA TGGCAGAGGG TGGCGTCCTT -637 GTGTTTCCTG CTGTGTTCAG TTCCTGGAAC AGTGCCCAGT ACCCAGGAGG TCCTTGGTCA -577 GGAGCTGAAG GAATTTTGCC CTCTAGTTTG GCAAGTTAGT GTTCCACATG ATTTCCCTAC - 5 1 7 ATGGAATTGT ACCGCCTCGG CCAAGGATGG TAGCGTGCCC ATGATCCAGA CTCGGGGGAG -457 ACTGAGGCAG GAGGTTCCTG AGTTCAAGGC TGGTCTTCAT AGCAAGACTC TCTCTGGAGA -397 AACTCAAATA AAACAAACTT TGAAAACATA AACTCTGATT GGTAAGAAAT AGCCAAGGCT -337 AGAGAGGATT CTAACCCTTG CTATTTGGTT GTCTTCCTTT CTCCATCATA GTCTCTCTCT -277 CTCTCTCTCT CTCTCTCTCT CTCCCTCATC CCTCCTTCTC CCTCCCTCTC TCCCTCCGTT -217 CCTCCCTCCC TCTTTCCCTC CCTCCTTTTT TGAGACTGCA TCTTACTATG TATGGAAGGG -157 GCAGAGTTTC TTGAACTATG GAAGAGGAAG GGCTGGGGGG GGGGGCGGGG GCGGGGCGGG -97 GGGAGGCGAG GCAACCCCTG GAGCTGACTC ACTCTTTAGC AAACAGTGGG AGGAGCCCTA -37 GGGTCACAAA ACTGTCTTCT TCCCCTCGGT C A G T C T P 24 ATGGGACTCC CAAGGCGGCT GCTGCTGCTG CTGTTGCTGG CGACTACCTG TGTCCCAGGT 84 MetGlyLeuP rOArgAirgLe ULeuLeuLeu LeuLeuLeuA 1aThrThrCy SValProA -4

GAAGAGGCGT CTCTCATCAA CTGCCGGGGA CCAATGAATC AGTGCCTGGT GGCTACAGGC 11844 GluGluAlaS erLeuIleAS nCYSArgGly PrOMetASnG lnCySLeUVa 1AlaThrGly TTAGATGGTG AGGCCCTGTG LeuAspV 228 AGACTGATTG ACAGATGGTC GGATGGTGGT ACACAGTTTA TTTGAGGACA GCCTGGTCCA

TAGAGGAGGA GGCTCCACGT TGCACAGTGC

2220 nucleatldes

TTAGAGAGAG GGAGTGGACC AGGCCTAGAT CAGTTTCACA ACTCCTCTCC TCTCCTCTCC

TGGCCCAGCG GGCACAGTCA CTGGCACACT

CTCCACCCTT 144 GACTCCCGCA 204 GGAGGACGCG 264

. . . . . . . . .

ATGGGCAATCAGGGCACAAT GCATGCATCC AAGGGGGAAC 2544 CCATGGTGGGGTGGGAGGCA GACCCTGGCC ATCACCTCTG 2604 TCTCCTCTCGCCCAGCCTCC CAGGGCCTGC AGTGCATGCA 2664 - 4 laser GlnGlyLeuG 1nCySMetGl SP

GTGTGAGAGT nCySGLuSer

AACCAGAGCT AsnGlnSerC

GCCTGGTAGA ySLeuValG1

GACTACCGTG gThrThrVa1

CTTCGGGAAT GGCAAGGTGA LeuArgGluT rpGlnA 34

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GGGGACCCAC

CGGTTGGATCTTCCTATGGT CTCCCAGCAC TTGGGAATCA TACAGAGAGGCCCTGTCTGG

TGAGGCACTG

AAAGGCGACA GAGGTAGGTG GGGGTGGGGA

11904 AGCGCTGATA

CTTTAGCTGG 11964 GATCTCTGGG 12024 GGAGGCGGTG 12084

420 nucleotides

AGTATCTCTT AAATCAATCC GCCCTGCTCC CTTTCCTGAG

- 2" 7

AAGAGCTGCG TGGTGGCACC GGGCGTGAGC CTATCCAGCT ATTCTCCGGG TGTGTTCTCA GGCATTCATT TAGTAAACGC TGATCCCTGG

GGAGCTGGAA

GTACACACAA AAAGCTTGGA AGGGCCAAAT GACATCTCAG TGTTACTATG 12564 TGGTGTGAGT CTGAGGCACT GCCAGGGGTT CCTGGAACAC ACTTTGGGAA 12624 CAACAGGGAA CTGGTGGTTG GCGATGGCGT CCACCACCAG GAGCCTGTCA 12684 AATTCTTACT CGGACTCACC GACTCTTCTT TTTCCTCAGT GCTGGGAAAC 12744 228 a ILeuGlyASn GCTTCCTGGT AlaSerTrpC

GCCAAGGCTC YSGlnGlySe

CCACGTGGCA 12804 rH15ValAla

CGGAGTTATA ArgSerTyrT

CCGTAAGAGG hrValArgGl

CTGCGCCACG YCySAlaThr

GACTCCTTCC ASpSerPheP

CGACCCACCT roThrHiSLe

12864 CAACGTCTCT GTCTCCTGCT GCCACGGCAG CGGCTGTAAC UASnValSer ValSerCVSC VSHiSGlVSe rG1VCVSASn "

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AGCCCCACAG GGGGCGCCCC CAGGCCAGGC CCTGCTCAAC TCAGCCTCAT TGCCTCTCTG 12924 SerPrOThrG 1yGlYAlaPr OArgProGly PrOAlaGlnL euSerLeuTl eAlaSerLeu

c

CTCCTGACCC LeuLeuThrL

GPI

TCGGACTATG euGlyLeuTr

GGGCGTCCTC CTCTGGACCpGlyValLeu LeuTrpThrE nd 304

12984

8 13044 GGCTGACCAC

CCCCCAACAA

CCGATAGCAA 2784

ACAATAGGCC AGGAAACCAT CTCTCTCCAG ATTGTAAAGA TATACAAAAG TTTAAACGTC 2844 TFJLCAAGGGT GGAGAGGCAG CCCGGCATTT AAGACCAGCC GCTCTTCTCT TCCAGAGGCC 2904 TGGAGTTCAG TTCAGGTAGC CAGCCAGATG GGGTGACTCA CAACCACCCA TGACCCCCAG 2964

. . . . . . . . . .

1 0 8 0 nucleotides

ATAACAGAGT GGGCTCAAGC AAGAAGAAAG GCTCACAAGG GTTGGCCCTGGAGATGAGTG 4104 ACTCTGCGGC CAGGAGTGTC ATGGGCTGGC TGGACCTTGG AGCTTCTGAG GGACTAAAGC 4164 TGTTTTCCTC ACAGATGATA GAGAGCTGGA GGTGGTGACA AGAGGCTGTG CCCACAGCGA 4224 3 4 SpAspA lgGluLeuG1 uValValThr ArgGlyCysA laHiSSerGl 4284 AAAGACCAAC AGGACCATGA GTTACCGCAT GGGCTCCATG ATCATCAGCC TGACAGAGAC uLySThrASn ArgThrMetS erTyrArgMe tGlYSerMet IleIleSerL euThrGluTh

CGTGTGCGCC ACAAACCTCT GCAACAGGCC rValCySAla ThrASnLeuC YSASnArgPr HI CCCTAGCCAA AGCCCATCAC CACCCCAATT TTCCTCACCC CGGCCCGAAT CCATCTATAG

cr

AGTGTCCCAA

GACATCTCCC

CAGACCCGGT GAGTCGGGCG OArgPrOG 82

TCCTTGCCAC 4344

CTATCCCCAG ACCTGCATTA CCCCAGTCTG 4404 CCCTACCCAA ATTTCACACC TGCCCCCATC 4464

TCCCCCATGT

CGAAATAGCC

GGACACCCAG

TGGTTTTGTT 4524

4 7 4 0 nucleotides

GATAAGTAGG CTTGGTGATT 9324 AAAGGCGGGA AAGGACAGGA TTCATTCTAG AAGGACTTGG CGCCAACACG CCACTCTCTC 9384 GGCTGCCAGG TTCAGAGTGG AGTTCTCTGC AGGACCTGGC GTTACCTCGA GTGTGCGTCC 9444 CTCTTCCTAG GAGCCCGAGG CCGTGCTTTC CCCCAGGGCC 82 1yAlaArgGl yArgAlaPhe PrOGlnGlyA rgTyrLeuGl uCySAlaSer b R2 TGCACCTCTT TGGACCAGAG CTGTGAGAGG GGCCGGGAGC AAAGCCTGCA ATGCCGCTAT 9504 CysThrserL euASpG1nSe rCySGluArg GlYArgGluG lnSerLeuGl nCysArgTyr CCTACAGAGC ACTGTATTGA AGTGGTGACC CTCCAGAGCA PrOThrGluH isCVSIleG1 UValValThr LeuGlnSerT

V

CAGAAAGTAA GCTCCCATCT 9564 hrGluA 134 SerLy ~LeuProSer

GCTGGGCAAC TCCTAGTAGA GATCTTCAAG TCCTGGGAGC AGAGCGCAAG CAAGAGACAA 9624 AlaGlyGlnL euLeuValG1 uIlePheLys SerTrpGluG lnSerA1aSe rLySArgGln CTGAATCCAC ACACAGTCAC GGGACCAACA LeuASnPrOH IsThrValTh rGlyPrOThr GATCAGCTTG AspGlnLeuG

TTCTCAGTGA CTGGAAGTTC CCGGTCACTG 9684 PheSerValT hrGlySerSe rArgSerLeu

9744 GGAGTGACCA GGAACCCAGC TACCTTATCA TGTCTCCCAT ATTGCTTTCG 1ySerASpGl nG1uPrOSer TyrLeuTleM e t S e r P r o T l ePheLeuSer

9864 TCCAGAGAGA AGAAAAGGGGAAAGAAGGGC CCCAAAATTC CTTTGTTTTG TAAGGGTTTT 9924 CTTTCTAGAA ATCCCAGAAACGCATAAGGA TCTATTTATC GTTCGGGCCT CCTTTATCTA 9984 TAACAAAGGG GAAAAGGAACACGAAGCCCT TTGGCTAAGG ATGAAGTTAC CCTAAGTAGC 10044 AAGGGCCTCT GATAAAAGGGGTGTGCGCAA GGGGGCAAAT CAACACCCAG CCCCTTACCA 10104 GGATCTGCCC TCTGCCTTCTGCAGGGAGCT TGAAGGATGA GGACTACACC CGAGGCTGTG 134 rgSerL euLySASpGl uAspTyrThr ArgGlyCysG

GCAGTCTTCC CGGATGCCCA GGCACAGCAG GTTTCCATAG CAACCAGACC TTTCACTTCC 10164 lySerLeuPr ~GlyCysProGlyThrAlaG lyPheHisSe rASnGlnThr PheHiSPheL TGAAGTGTTG CAACTACACC CACTGCAATG GTGGCCCAGG TGAGGAGCAG GAAAGATGAA 10224 eULySCySCy SASnTyrThr HiSCySASnG lyGlyPrOV 179 RZ 10284 GTGGACATTC CTGTCTCTTT CTACAACTCT CTCTCCTCAC TCTCTCCTAA GTCCGTTCAA ACAGCATAAG TATCGCCTCT CCTGGAGGTC TGCCCTGACT TGCAAATCTG CTACTGAGTC1 0 3 4 4 10404 ACTCAATTCT CCATGCCCCA GCTCTGCCCA CTCTGGGTGG GGACTGTCTG CTGACAAAGC

cr

. . . . . . . . . .

1 1 4 0 nucleotides

. . . . . . . . .

GAGAACCAAA TGCCAAAAGA ATGCAGTTCC TGGGCATGAC CATCAGGTGG CCTCAGGCTG AGTGTGGGCA GATGGATGGC TGCATAGACC CAGTGCTCAT TGTCGTTTTT TTTTTTTTTT TTTTTTTTTC CATCTTTAAG TTCTGGATCT 1 7 9 alLeuAspLe

CGCCCTATCA 1 1 6 0 4 CTTCGTTCCC 11664 TCAGAGCTTT 1 1 7 2 4 uGlnSerPhe

11784 CCACCGMTG GCTTCCAGTG TTACAGCTGT GAGGGGAACA ATACCCTTGG GTGTTCCTCC ProProAsnG 1yPheGlnCy sTyrserCys GlUGlyAsnA SnThrLeuGl yCysSerSer

FIG.2. Nucleotide sequence of the murine UPAR gene and its 5'- and 3'-flanking regions. The proposed site of transcriptional initiation is defined as nucleotide +l.The site of polyadenylation is nucleotide 13,207 based on the reported muPAR2 cDNA sequence (33). The 876 nucleotides of the included 5"flanking sequence are numbered -1 to -876. The numbers to the right of each line correspond to the last nucleotide of that line. The noncoding portions of exons 1, 4 (uPAR splice variant), and 7 are underlined. The deduced amino acid sequence is shown below the protein coding portions of the exons. The location of repeat ( R ) domains, the signal peptide (SP), and the putative glyco-

13104 13164

~

GTGAACAAGG AATTGTTTTT ATCTCACCAT CTCACAGAGA

AAAAGAT TTTTGTACCA1 3 2 2 4 TTGGGTCTCC CCACTGTCAC ATTTAGCTTG GGAAGATGAG AGCAGCAGGG 13284 TTGTTTGTTTGTTTGTTTGA TTGATTGATT GATTTTTGCT TTGAGACAGG 13344 GTAGCCCTAGCTGCCCTGGA ACTTACTTTA TAGACCAGGC TAGCCTAAAA 13404 CCCACCTGCCTCTACCTCCT CAGTGCTTTG ATTAAAGGTG TGTACCACCA 13464

FIG.2"continued

recognized on the basis of cysteine placement, and a GPI anchor. uPAR is unique among the known Ly-6 superfamily members in that the protein contains three cysteine-rich domains, with the NH,-terminal-most repeat primarily responsible for uPA binding (41). Consistent with a modular form of gene evolution based on exon shuffling (42-441, the exon boundaries of the murine uPAR gene delineate the functional protein domains. The signalpeptide is largely encoded by the first exon. Internal cysteine-rich repeat 1, which is the uPA-binding domain (411, is encoded by the second and third exons. Exons 4 and 5 specify the second internal repeat.Exons 6 and 7 specify both the last internal repeat and the GPI anchor attachment sequence (45). A number of features of the murineuPAR gene are consistent with the view that it is a member of the Ly-6 superfamily. First, like Ly6C.l and CD59 genes, which encode a single cysteinerich domain, each cysteine-rich domain of the uPAR gene is encoded by two exons. Second, the signalpeptide is encoded by a n independent exon. Third, the glycolipid anchor attachment domain is encoded by a single exon together with the COOHterminal portion of the final cysteine-rich repeat. Finally, like the Ly6C.1(28) and CD59 genes (291, all six splice junctions are type I (461, with the introns occurring between the first and second nucleotides of codons. Unlike the otherknown members of the Ly-6 superfamily, the murine uPAR gene does not have an entirelynoncoding first exon based on uPAR mRNA primer extension studies and the uPAR cDNA sequence (see below). Notably, the human uPAR gene also appears tolack a noncoding firstexon.' Based on the sequence organization of the murine uPAR gene, as well as the sequence and distribution of protein domains within exons of the Ly-6 family genes, it appears that the UPAR gene evolved secondary to two internal duplication events within a Ly-6-like ancestral gene as illustrated in Fig. 3. Exon Sequence Polymorphisms in the uPAR2 Splice Variant-Comparison of the murineuPAR gene sequence with the published murine uPAR cDNA sequence revealed five difE. Soravia and F. Blasi, personal communication. sylphosphatidylinositol attachment site (GPI)are indicated by the arrows. The two oligonucleotides used in primer extension studies (see text) were complementary to the exon 1 sequence numbered 5 P 8 1 and the exon 2 sequence numbered 2649-2678. The complete sequence, including complete intron sequences, has been deposited inthe GenBank data base.

Urokinase Receptor Gene

blocks. Repetitive C+T-rich (or A+G-rich) sequences are observed in the 5'-proximal region of many different genes, in-

TABLE I Sizes of the exons and introns Exon

Size

Intron

25995

Size

Intron type-

bp

1 2557 1 82 2 1428 2 111 3 144 3 5072 4 156*518b (374") 4 (300') 5 135 5 1501 882 6 147 6 Total intron 11,958b (11,740') 7 474d Total exon 12496.d (1467'~~) Total gene 13,207bd

I I I I 1 I

cluding the murine uPA (39), rat thyrotropin receptor (531, Drosophila hsp26 (54), and human TGF-P3 (55) genes. This interesting motif is has since it shown been that polypurine elements may adopt a non-B-DNAconformationhypersensitive to S1 nuclease digestion (56, 57). Furthermore, DNA-binding proteins that bind to TCCCTC sequence blocks have been described. A CCCTC-binding factor hasshown been to act as a negative regulatory transcription factor when bound to the proximal promoter region of the chicken c-myc gene (58). In contrast, the 10-fold greater TGF-P3 mRNA observed levels in the A375 melanoma cell line over other tumor cell lines have been attributed to a novel transcriptional transactivator that binds to the repeated TCCC motif within the TGF-p3 proximal promoter (59). The function ofthis sequence in the uPAR gene,

a Intron type according tonomenclature proposed by Sharp (46) with type I introns occuning between the first and second nucleotides of a codon. ThePredominantGPI-anchored splice variant, muPAR1, as described by Kristensen et al. (33). e The splice variant, muPAR2, which lacks the GPIanchor (33). if has not yet been d Exon size based on the known polyadenylation site defined pre+ A third class of repetitive element identified was the Alu-like ously in muPAR2 (33). I11 Type repetitive sequence, also referred to as identifier se-

ferences, all within exon 4. The cDNA nucleotide residues T347, A575,G606, G651, and G661(in the numbering system of Ref. 33) were found to be Cg417,GgM5, C9676, A9721, and C9731in the corresponding gene sequence, respectively. The lastthree of these apparent polymorphisms result in changes in the predicted amino acid residues: Gly176, vall~l,and k g 1 9 4 in the cDNAare Arg, Ile, and Pro in the gene, respectively. Notably,all of these amino acid changes occur withina region of exon 4 that occurs only inthe mRNA for therare alternative splice variant muPAR2 (33, 47). The uPAR2 splice variant is intriguing in that the deduced protein sequence would predict a soluble form of the receptor that includes the amino-terminal uPA-bindingdomain, but lacks the carboxyl-terminal glycolipid anchor attachment site. However, the uPAR2 protein, unlike the common uPARl form, contains an odd number of cysteine residues and therefore, may be unstable. Indeed, the protein correlate of the murine uPAR2 mRNA splice variant has not yet been found, and the uPAR2 protein synthesized by cultured cells transfected with uPAR2 cDNA expression vectors is not successfully ~ e c r e t e dThere.~ fore, the many apparent polymorphisms resulting in amino acid differences between the published uPAR2 cDNA and the presently reported uPAR gene couldreflect a lack of functional constraint in this portion of the gene which may not encode a functional protein. In this regard, it is notable that the splice junction resulting in the uPAR2 transcript conforms the least t o the consensus splice junction proposed by Mount (40). Repetitive DNA-Several different repetitive sequences were observed in the introns and flanking regions of the murine uPAR gene (Fig. 4). B-type repeats (48-51) were the most frequently observed class of repetitive elements. B1 elements were foundin introns1,2,3, and 6, as well as in the 3"flanking region, while B2 elements were present in introns 1 and 5. As observed in themurine uPA gene (391, many of the Belements were partial B repeats and consequently, lacked part or all of the consensus RNA polymerase I11 bipartite promoter present in the complete B elements. Of these B-type repetitive elements, the B1 repeat in the 3'-flanking region could have a potential functional significance in that contains it an ATTAAA box, which could be used as a possible alternate polyadenylation signal (51, 52). Another class of repeat observed in the murine uPAR gene consisted of a polypyrimidine (or polypurine on the complementary DNA strand) sequence. Starting at -305, there was a C+Trich stretch of sequence containing primarily CT and CCCT K. Dane, unpublished observations.

quences (60).At the end of intron 3from +8423 to +8867, there were a series of eight 304,~direct repeats conforming to the general sequence: 5' -TCCCCCAGTGA(G/A)GGGCTGGGGGCGTGGCT(C/T) - 3'

alternating between nine 23-bp direct repeats of the general sequence: 5' -AGTGGTAGAG(C/T)CCCTGCCTAGAA - 3'

Within the 30-bp motif were two 7-bp inverted repeats (indicated by the underlined portions above). Identifier sequences, which have a high (>go%)sequence similarity, are significant in that they have been suggested t o be involved in tissue-specific gene activation (60). Finally, there was a G+T-rich sequenceblock within intron 3 (+5916 to +6197). This approximately 280-bp region was primarily composed of an alternating purine-pyrimidine homocopolymer of the form (TG),. Althoughsequences of this kind are commonly found in eukaryotic genomes, they are notable because they can lead to left-handed Z-DNA structure (61, 62). Site of Znitiation of Dunscription-The site of transcription initiation was determined by primer extension analysis using two different synthetic oligonucleotides complementary to regions in exon 1 and exon 2 (see "Experimental Procedures"). Oligonucleotideswere end-labeled, hybridized to poly(A)+ mRNA isolated from phorbol 12-myristate 13-acetate-stimulated J774A.1 cells, and extended with reverse transcriptase. Fig. 5 shows the major primer extension products from the endlabeled oligonucleotide complementary to exon 2 sequences indicated in Fig. 2.A major primer extension product of 120-122 nucleotides in length was detected in mixtures with J774A.1 1 )but not in mixtures with irrelevant conpoly(A)+ mRNA(1ane trol RNA (lane 2 ). Using the exon 1oligonucleotide(see Fig. 2), the major primer extension product was about 81 nucleotides in length (data not shown). In both cases, these data indicate that the major transcription initiation site in J774A.1 cells is at or near the nucleotide labeled +1in Fig. 2. Importantly, all of the nucleotides assigned as 5'-noncoding sequence based on these primer extension studies were also foundwithin the nucleotide sequence of the uPARl cDNA (data not shown). 5' End ofthe Murine uPAR Gene-The 5'-proximal region of the murine uPAR genedoes not contain obvious TATA and CAATbox motifs, features that may result in the observed imprecision in theselection of the transcription start site. However, consistent with many vertebrate genes transcribed by RNA polymerase 11, the murine uPAR gene has an upstream GC-rich island. A G+C-rich 40-bp regionwith a base composi-

25996

Urokinase Receptor Gene TABLE I1 Intron splice junctions Donor match to consensus"

Intron

1

Exon

3 4

5 6

Acceptor

CCAG ZAAGa TCTCCTCTCgCCCAz CAAG EGAGg CTgTTTTCCTCACE CCCG GTGAGT...CTCTCCTCTTCCTAG GAAa EAAGc CTCTgCCTTCTGCE CGATcA a tGg. ..CTCTgCCTTCTGCAG CCAG EGAGg TTTTCCaTCTTTaAz GAtG GTGAGg CTTCTTTTTCCTCAG C G ccccccccccc cAG /GT AGT.. N AG/ A TTTTTTTTTTT T

14/16 14/16 16/16 15/16 15/16 13/16 15/16

cCTC aTGA GAGC GGAG GGAG tTCT tGCT

... ... ... .

A

match to consensus"

Exon

... ...

819 819 W9 7/9b 519" 819 719

2

Intron

G

Mount consensus

The fraction of nucleotides conforming to the Mount consensus sequence (40).

* Alternate splice site of the predominant mRNA species, muPARl(33). Alternate splice sites of the other mFtNA species, muPAR (33).

Ly6C.l Gene

-

SP

GPI

"Repeat"

0.5kb

n

n

&?

I

Murine uPAR Gene

lll--cc-8ssc

rr

I

H

I

u

f

Repeat 1

SP

I

I

U

Repeat 2

-?

Repeat 3 fNc GPI

FIG.3. Comparison of the gene organizationof Ly6C.l and uF'AR.. Exons are indicated by wide bars with regions corresponding to the cysteine-rich internal repeat of uPAR bracketed and dark-stippled. The regions coding the signal peptide (SF')and glycolipid attachment motif (GPZ) are shown as filled and light-stippled areas, respectively. The noncoding portions (NC) of exons are shown as open areas. 0.5kb

EXON 1

EXON 2

+

B2

0

(CT),

D

EXON 3

D

EXON4

D

EXON 5

EXON 6

EXON7

D +m

D

B1

Nu-like Type 111

(Grin

FIG.4. Repetitive elements within the uPAR gene. The location and relative size of various repeat sequences found in the uPAR gene are illustrated below the gene map. The arrowheads on the B family and Alu-like Type I11 repeats indicate the orientation relative to putative split polymerase I11 promoter elements.

tion of 92.5% G+Cis located between-126 and -87. A survey of the restof the gene revealed that theoverall G+C content was 49.9% and that no other 40-bp region exceeded 77.5% G+C. Furthermore, a perfect match to the 10-bp Spl consensus sequence, nested within three perfect matches to the 6-bp Spl core consensus sequence, is located within this G+C-rich region in the proximal promoter (Fig. 2). It is also notable that the

G+C-rich region contains the 9-bp sequence GCGGGGGCG, which is a perfect match to the canonical binding site for the Krox-20 family of transcription factors, including NGFI-A and NGFI-C (63). A number of other potential regulatory features were observed in this areaof the gene. Betweenthe putative Spl binding sequence and the transcription start site, there isa possible

Urokinase Receptor Gene I 2 G A T C -

l40b +

121b

lOOb +

Flc. 5. Determination of the site of transcription initiation by primer extension analysis. :y2P-Labeledoligonucleotide primer complementaq to the region numbered 2649 to 2678 (exon 2 ) in Fig. 2 was hybridized to either 1 5 pg of yeast tRNA (lane 1 ) or 15 pg of poly(AY RNA isolated from J774A.1 cells (lane2). Reverse transcriptase-generated primer extension products were displayed on a DNA sequencing gel alongside a sequencing ladder generated from a 390-bp EcoRIIXbaI fragment from the 3'-noncoding flank of the murine uPAR gene (Fig.1). The relative position of the 121 nucleotide primer extension product is indicated by an arrowhead. For reference, the relative positions of 100 and 140 nucleotides on the sequencing ladder are shown by arrows.

AP1 binding site (-72 to -66). In addition, there is a potential PEA3-like regulatory sequence (-135 to -126) upstream of the Spl consensus sequence match. These particular regulatory elements arenoteworthy given the well documented induction of uPAR expression by tumor promoting phorbol esters (64-66) and the known linkagebetween AP1 and tumor promotermodulated gene expression (67, 68). It is interesting to note that a PEA3 element inconjunction with anAP1 site within an enhancer has been found t o be involved in the phorbol ester induction of the human uPA gene (691, and both sites may contribute to cell type-specific expression (70). Similarities between the regulatory elements of the uPA and uPAR genes could provide a basis for coordinating expression of these proteins. However, while many cell types produce both uPA and uPAR, there are manyexceptions such as mouse LB6 cells (71) and humancolon adenocarcinoma cells (72) thatexpress uPAR but express little or no uPA. The availability of the cloned and fully characterized murine uPAR gene shouldbe a valuable tool in genetically definingthe regulation and biological role of surface-associated plasminogen activation. In particular, the ability to functionally ablate or alter theuPAR gene in transgenicmice makes itpossible to address the role of uPAR embryonic development and in processes such as fibrin clot lysis and tumor cell invasion and metastasis. Acknowledgments-We thank Drs. T. Doetschman and M. Shull for the 129/SvJ strain mouse genomic DNAlibrary. We gratefully acknowledge N. Jensen and M. Flick for technical assistance. We also thank Drs. M. Ploug, T.Bugge, and K. Holmback for their helpful suggestions. Note Added in Proof-While this paper was in press, a partial sequence of the human UPA receptor gene was reported (73). REFERENCES 1. Collen, D., and Lijnen, H. R. (1987) in The Molecular Basis ofBlood Diseases (Stamatoyannopoulos, G., Nienhuis, A.W., Leder, P., and Majerus, P.W.,

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