The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential applica- tions in interfering with cell-surface plasmin-mediated.
Vol. 266, No. 12, Issue of April 25, pp. 7842-7847, 1991 Printed in U.S A .
THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ligand-binding Domain of the Cell Surface Receptor for Urokinase-type Plasminogen Activator* (Received for publication, October 25, 1990)
Niels BehrendtSB, MichaelPlougS, Laszlo PatthyV, Gunnar Houenll, Francesco Blasi**, and Keld Dan& From the $FinsenLaboratory, Rigshospitalet, Strandbouleuarden 49, DK-2100 Copenhagen 0..Denmark, the BZnstitute of Enzymology, Siological Research Center, Hungarian Academy of Sciences, H-1502 Budapest, Hungary,and the Institutesof IIBiochemical Genetics and *‘Microbiolom. Universitv -” ” of. Copenhagen, 0ster Farimagsgade 2A, DK-1353 Copenhagen K., Denmark ,
The purified urokinase plasminogen activator receptor (u-PAR) was cleaved into two fragments by mild chymotrypsin treatment. The smaller fragment (apparent M, 16,000) possessed the ligand-binding capability, as shown by chemical cross-linking analysis. This fragment constituted the NH2-terminalpart of the intact receptor, probably including the whole sequence 1-87, and contained N-linked carbohydrate. After detergent phase separation in the Triton X-114 system, the fragment was present in the water phase where its binding activity could be demonstrated in the absence of the rest of the protein. An analysis of internal homology in the amino acid sequence of u-PAR revealed the presence of three repeats of approximately 90 residues each. The ligand-binding fragment corresponds to the first repeat, supporting that this unit is a structurally autonomousdomain. Domains homologouswith the internal repeats of u-PAR constitute the extracellular part of Ly-6 antigens and of the squid glycoprotein Sgp-2. Like u-PAR, these proteins are attached to the membrane by a glycosyl-phosphatidylinositol anchor. The hydrophilic, ligand-binding u-PAR domain identified in the present study has potential applications in interfering with cell-surface plasmin-mediated proteolysis.
participation in the protein degradation processes occurring during tissue remodeling, and cell invasion under normal and pathological conditions, including cancer (1-3). Plasminogen activation is subject to stringent regulation involving, among other factors, the specific localization of uPA to cell surfaces. u-PA and its proenzyme, pro-u-PA, bind to a specific receptor protein (u-PAR) or certain cell types, including a number of cell lines of neoplastic origin (4-11). On certain cell types, this receptor has been shown to localize u-PA to cell-cell and focal cell-substratum contact sites (12). In addition, the binding of pro-u-PA or u-PA to uPAR, in conjunction with cell surface binding of plasminogen to as yet unidentified binding sites, leads to a strongly preferential plasminogen activation occurring on the cell surface (13, 14). The receptor-binding part of u-PA is located in the amino terminal fragment (ATF) of the u-PA A-chain, constituting amino acid residue no. 1-135, the sequence 12-32 being important for binding (15). The isolated ATF competes with uPA for binding to u-PAR and can be used directly, in the radiolabeled form, to monitor the ligand-binding activity of u-PAR (5,7). The receptor binding capability is independent of the catalytic site of u-PA (4,5, 16). Up to now, no information has been available concerning the structures within u-PAR that govern the ligand binding activity. We have previously reported that thehuman u-PAR can be purified by affinity chromatography; it is a highly The urokinase-type plasminogen activator (u-PA)’ is one of the two known specificenzymes, capable of converting the glycosylated protein, consisting of a single polypeptide chain, proenzyme plasminogen into the broad specificity protease which exhibitssubstantial M , heterogeneity andvariation plasmin. Plasmin is a key component in extracellular prote- among different cell types due to differential glycosylation (7, olysis, being active in a variety of processes which include the 17). The amino acid sequence of u-PAR is known due to degradation of extracellular matrix and basement membrane sequencing of its complete cDNA (18); the mature protein is proteins, the activation of pro-collagenases and pro-forms of carboxyl terminally processed and membrane attached by a growth factors, and thrombolysis. While plasminogen occurs glycosyl-phosphatidylinositol anchor (36). These results proat a high concentration in the extracellular fluids, active vide the background for the present study, in which we report plasmin is formed only under very specific conditions, and the identification and isolation of the ligand-binding domain plasminogen activation is a central event in the reaction route of u-PAR and demonstrate that this domain can exert its leading to the above processes. An important role of u-PA is biological activity autonomously, ina water-soluble form. This finding is in accordance with studies on internal and *This workwas supported by the Danish Cancer Society, the external homologies of u-PAR predicting that the identified Danish Biotechnology Program, and the Danish Medical Research ligand-binding fragment corresponds to a structurally indeCouncil. The costs of publication of this article were defrayed in part pendent domain in the receptor molecule.
by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 EXPERIMENTALPROCEDURES solely to indicate this fact. Preparation of Purified u-PAR-Purification of u-PAR by temper$ To whom correspondence should be addressed. ’ The abbreviations used are: u-PA, urokinase-type plasminogen ature-dependent Triton X-114 phase separation of cell extracts folactivator; u-PAR, u-PA receptor; ATF, amino terminal fragment of lowed by affinity chromatography on immobilized diisopropyl fluou-PA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-l-pro-rophosphate-treated u-PA was performed as described (17), except panesulfonate; DSS, N,N’disuccinimidyl-suberate; SDS-PAGE, so- that the raw material was phorbol 12-myristate 13-acetate-treated U937 cells of the variant cell line previously designated U937a (17, dium dodecyl sulfate polyacrylamide gel electrophoresis.
7842
Ligand-binding Domain of u-PA Receptor
7843
obtained from Dr. A. Fattorossi, Research Laboratory of Aeronautica sequences for the internal repeats were performedaccording to a Militare, Rome, Italy). Allof the protein detectable in the purified described method (23). The National Biomedical Research Foundation/Protein Identification Resource database was searched for hopreparation by SDS-PAGE and silver staining was able to bind to nonlabeleddiisopropyl fluorophosphate-treated u-PA or ATF in a mologous sequences usinga proceduresuitable for detection of distant chemical cross-linking assay (seeRef. 17 for details on this analysis). homologies (23-25). Inthis procedure, similarityto aconsensus sequence characteristic of a protein family is used to decide whether EnzymaticDigestion-Chymotrypsin (67units/mg)andtosylphenyl chloromethyl ketone-treated trypsin (255 units/mg) were from a test protein has the featurestypical of that protein family. Worthington Biochemical Corporation,Freehold,NJ.Endoproteinases Glu-C and Lys-C (sequencing grade) and bromelain were RESULTS from Boehringer Mannheim, Federal Republic of Germany. Search Liberation of a Ligand-binding Fragment by Proteolytic for degradation conditions was carried out by overnight incubation of purified u-PAR (3 pg/ml, used directly in the form of neutralized Digestion of u-PAR-In order to study the structural features affinity column eluate (17)) with eachof the proteases, added in the of u-PAR importantfor the ligand binding capability, samples form of a dilution series (final concentrations ranging from 8 ng/ml of the purified receptor were treated with dilution series of to 1 pglml, or to 0.2 pg/ml in the case of Lys-C). For systematic studies on degradation with chymotrypsin, purified u-PAR was con- various proteases under nondenaturing conditions. The dicentrated by dialysis and lyophilization as described (17) and redis- gested samples were analyzed for binding activity,using chemsolved a t 30 pg/ml final concentration in 0.05 M Tris/HCl, 0.05% ical cross-linking to theradiolabeled ligand (7). CHAPS, pH 8.1, followed by addition of chymotrypsin (40 ng/ml, or Of the five proteasestested (see "Experimental Proceas indicated). After incubation for 7 h at 37 "C, the degradation was dures"), chymotrypsin proved abletogenerate a distinct, stopped by addition of 1 mM phenylmethylsulfonyl fluoride (Merck, ligand-binding fragment of u-PAR, while none of the other F. R. G.), added in the form of a fresh 20 mM stocksolutionin enzymes had this capability. The formation of the fragment dimethyl sulfoxide. Chemical Cross-linking Assay for Ligand-binding Activity of u-PAR was evidenced by the observation of an approximately M, and u-PAR Fragments-ATF (amino acid residue no. 1-135 of human 32,000 radiolabeledconjugate after cross-linking to ""I-laurokinase) wasa kind giftfrom Dr. G. Cassani,LePetit, Italy. beled ATF (Fig. 1, lune 3 ) . This component was absent in Chemical cross-linking of intact or degraded u-PAR to '2511-labeled those samples where the intact receptor was tested (lane Z ) , ATF, using N,N'-disuccinimidylsuberate (DSS), was performed as showing only the M, 70,000-80,000 conjugate described predescribed (17). Visualization of the formed cross-linked conjugates was performed by SDS-PAGE according to Laemmli (19) on gradient viously for the intact u-PAR(17). The intensity of the latter conjugate was strongly reduced in the chymotrypsin-treated slab gels (6-1696 polyacrylamide), followed by autoradiography. In some experiments, nonlabeled ATF wasused as the ligand. samples, reflecting a rather efficient cleavage of the active These experiments were performed in the same manner, except that receptor (see also below). No other ATF-binding products the protein concentrations were those indicated and that electropho- were detected. The electrophoretic patterns were identical, retic analysiswas performed by Tricine-SDS-PAGE andsilver stain- whether SDS-PAGE was performed with reduced or nonreing (see below). HydrophobicityAnalysis of duced samples (compare Fig. 1,A and B ) . Triton X-I14 PhaseSeparationfor Directanalysis of the chymotrypsin-treated samples by ATF-binding Components-Intact or chymotrypsin-treated u-PAR was diluted in 0.1 M Tris/HCl, 1%Triton X-114, pH 8.1, a t 0 "C and SDS-PAGE and silver staining revealed a very simple cleavincubated for 5 min a t 37 "C. The resulting detergent- and waterage pattern (Fig. 2A). The intact u-PAR, purified from the phases, respectively, were separated by centrifugation. Each phase present source, migrated asa heterogeneous component, covwas made up to the startingvolume by addition of 0.1 M Tris/HCl, ering theM,50,000-65,000 region (lune 3 ) ,this heterogeneity pH 8.1, after which CHAPS (0.25% final concentration) was added in order to avoid renewed phase separation. The presence of ATF- being due to variations in N-linked carbohydrate (17). The binding components in each phase was analyzed by chemical cross- chymotrypsin treatment led to the appearance of one major fragment of M , 16,000, which migrated as a sharp band, and linking t o 9 - A T F(see above). Deglycosylation Analysis-For enzymatic deglycosylation, samples a heterogeneous component in the M, 35,000-50,000 range, cross-linked to '2sI-labeled ATF were 1.5-fold diluted and denatured by boilingfor 3 mininthe presence of 0.5% SDS and 1.7 mM B A dithiothreitol. The denatured sampleswere further 6-fold diluted by addition of a deglycosylation buffer, to include final concentrations M,x~o-~1 2 3 ~ ~ ~ 1 01 - 32 3 of 0.12 M sodium phosphate, 0.9% Triton X-100, 5 mM lJ0-phenanthroline, and 33 units/ml peptide:N-glycosidase F (N-glycanase; Genzyme, Boston, MA), respectively, pH 8.6. lJ0-Phenanthroline wasadded in the form of a 250 mM stock solution in methanol. 94Deglycosylation was performed by overnight incubation of the sam94ples a t 37 "C. 67Electrophoretic Techniques for Fragment Analyses and Isolation of 67Components for Amino Acid Sequencing-Samples of degraded uPAR for amino acid sequence analysis were concentrated by lyophi43 43 lization before electrophoresis. Samples for gels to be silver stained were analyzed directly. 3030Tricine-SDS-PAGE according to Schagger andvon Jagow (20) was performed as described (17), except that 10% polyacrylamidegels were used. Silver staining was carried out using the reagent system of Heukeshoven and Dernick (21). Electroblotting of samples onto polyvinylidenedifluoridemembranes for amino acid sequencing was performed as described (22), FIG. 1. Detection of a ligand-binding u-PAR fragment by except that the concentrations of methanol and dithioerythritol in chemical cross-linking. Purified u-PAR (30 pg/ml) was treated thetransfer buffer were 15% and 0.5 mM, respectively, and that blotting was performed for 90 min a t a current density of 0.4 mA/ with chymotrypsin (40 ng/ml) for 7 h a t 37 "C (lane 3 ) , or analyzed directly, without degradation (lane 2). The control (lane I ) included cm2. No alkylation was performed. conditions. Amino Acid Sequencing-NH2-terminal amino acid sequencing was buffer, incubated with chymotrypsinunderthesame Analysis was performed by incubation of the 67-fold diluted samples performeddirectly on excisedpieces of polyvinylidenedifluoride membranes, containing electroblotted protein bands (above).An Ap- with "'I-labeled ATF (1 nM) and cross-linking with DSS, followed by SDS-PAGE under reducing (A) or non-reducing ( R ) conditions plied Biosystems protein sequencer, model 477A, was used. Alignment and Homology Analyses-The search for internal re- and autoradiography of the gels. The electrophoretic mobilities of peats in u-PAR, the construction of multiple alignment and consensus molecular mass marker proteins areindicated. ,
,
Ligand-bindingDomain of u-PA Receptor
7844
firmed that the large fragment (i.e. the Mr 35,000-50,000 component) was indeed devoid of ligand-binding capability. ~ ~ ~ 1 0 1- 3 2 3 4 5 ~ , ~ 1 0 - 31 2 3 4 While the latter fragment was unchanged onthe silver-stained ".' 200-. 200 gel also after treatment with the highest chymotrypsin con116. 11694 94' centration tested (i.e. 1 pg/ml), such samples were unable to 6767. form the M , 70,000-80,000 cross-linked conjugate with '*'I43 Y 43. ATF observed above when using lower amounts of the en' n zyme. 30 30. Identification of the Ligand-binding Fragment-Approxi20 .. -_ 20, mately 20 pg of purified u-PAR was treated with chymotrypsin 14'I (40 ng/ml) under the same conditions as used above. The 14. sample was concentrated by lyophilization and subjected to Tricine-SDS-PAGE, after which the gel was electroblotted FIG. 2. Electrophoretic analysis of u-PAR fragments. A, non-degraded, purified u-PAR (0.4 pg, lane 3), purified u-PAR (0.4 onto a polyvinylidene difluoride membrane. The membrane was stained with Coomassie Brilliant Blue, and the following pg) treated with chymotrypsin as described in the legend to Fig. 1 (lane 4 ) , or buffer treated with chymotrypsin under the same condi- stained areas were excised (see arrows in the parallel electrotions (lane 2 ) were analyzed by Tricine-SDS-PAGE under reducing phoretic pattern shown in Fig. 2 A ) : I,the M , 16,000 cleavage conditions.The gel was silver-stained. The molecular masses of product; II, the stained areacorresponding to the M , 35,000marker proteins (lanes 1 and 5)are indicated.R, purified u-PAR was 45,000 region, i.e. the region containing the larger cleavage degradedwith chymotrypsin as described in the legend to Fig. 1. Samples of the degradation mixturewere subjected to chemical cross- product, excluding the residual undegraded u-PAR. The excised polypeptides were subjected to NHderminal linking with DSS in the absenceof ligand (lane 2 ) or in the presence of ATF (lane 3 ) . Lane 4 shows the ATF preparation alone, after amino acid sequencing to yield the sequences shown in Table chemical cross-linking. Cross-linking was performed in a volume of I. Only one sequence was found in each case. The M , 16,000 12 pl, and each sample contained 0.3 pg of u-PAR material and/or fragment had the sameNH, terminus as thatof the intactu0.5 pg of ATF. The DSS-treated samples, and a 0.3-pg sample of uPAR degradation mixture which had not been subjected to cross- PAR, determined previously (17), while the larger cleavage linking (lane I ) ,were analyzed by Tricine-SDS-PAGE underreducing product had NH2 terminus at residue no. 88 of the intact conditions, followed by silver staining. The electrophoretic mobilities protein, as identified by comparison with the complete amino of molecular mass marker proteins are indicated. (18). acid sequenceof u-PAR, deduced from cDNA sequencing Hydrophilicity and Dissociation after Cleauage-Temperawhich appeared to represent the restof the molecule (i.e. the ture-dependent Triton X-114 phase separation (26) has been phase (17, starting materialdegraded by the removal of an approximately shown to direct the intact u-PAR to the detergent to thepresence of a COOHM , 16,000 fragment) (lane 4 ) . In addition, a small amount of 27), this hydrophobicity being due intact u-PAR was observed in this sample, consistent with terminal glycosyl-phosphatidylinositol glycolipid membrane anchor (36). In order to analyze whether the ligand-binding the residual ligand-binding activity observed above. This patternsuggested that theligand-binding u-PAR deg- u-PAR fragment would be able to dissociate from the rest of radation product was identical to the M , 16,000 fragment, the molecule under nondenaturingconditions, chymotrypsinsince thismolecular weight is in agreementwith the formation treated u-PAR was subjected to detergent phase separation. of an M , = 32,000 conjugate with ATF (apparent M , 18,000). The resulting phases were analyzed in the "'I-ATF crossAlternatively, however, the cross-linking activity could be due linkingassay (Fig. 3). The radiolabeled conjugate of M , t o a trace fragment undetectedby silver staining. To test this 70,000-80,000 (i.e. the adduct of "'I-ATF and the residual, intact u-PAR after digestion) was formed almost exclusively possibility, a cross-linking experiment was performed with nonlabeled components using silverstaining afterelectropho- in the detergent phase, while the M , 32,000 conjugate was observed only in the water phase (compare lanes 2 and 3). resis (Fig. 2B). It is seen that the electrophoretic appearance of the chy- The former observation provided an internal control of the it that theCOOHmotrypsin-treatedsample (lane 1 ) was unaffected by the phase separation, and furthermoreassured performance of the cross-linking procedure when no ligand terminal, hydrophobic character had not been lost as caused by e.g. contaminating phospholipases or undetected proteowas added (lane 2). On the other hand, when cross-linking was performed in the presence of ATF (lane 3), a unique M , lytic attack. The exclusive occurrence of the ligand-binding indicated that this 32,000 product was formed, while the Mr 16,000 band almost fragment in the water phase therefore disappeared. Some staining was observed in the M , 18,000- fragment had dissociated from the COOH-terminal, glyco20,000 region in this sample, but this was due to the surplus lipid-containing part in theabsence of denaturing agents. Glycosylution-u-PAR contains large amounts of N-linked of added ATF (lane 4 ) . Thus, the M , 32,000 conjugate was clearly detectable by carbohydrate which can be removed by treatment with the silver staining, and since its formation was accompanied by enzyme peptide:N-glycosidase F (17). When samples containconsumption of the M , 16,000 fragment, thiscleavage product ing l2'1-ATF cross-linked tochymotrypsin-treated u-PAR were treated with this enzyme (Fig. 4), theMr 32,000 conjugate was directly responsible forthe ligand-binding activity. Degradation of u-PAR using higher concentrations of chy- was deglycosylated to yield a conjugate of approximately M , motrypsin (0.2-1 pg/ml) led to the appearance of an additional 25,000 (compare lanesA2 andB 2 ) . The conjugate formed with silver-stainable fragment which migrated as an M , 13,000 intactu-PAR (lane A I ) was converted to the Mr 50,000 product (lune Bl ), described previously (17). Thus, theligandcomponent in SDS-PAGEunder reducing conditions (not shown). The use of a chymotrypsin dilution series indicated binding u-PAR fragmentis glycosylated. that this product arose from further degradation of the M , DISCUSSION 16,000 fragment. However, the Mr 13,000 product proved An M , 16,000 chymotryptic fragment of u-PAR was generunable to bind to "'I-ATF in the cross-linking assay, thus indicating that the activity of the ligand-binding fragment ated which, at least qualitatively, retained the binding capawas lost by this cleavage. In addition, this experiment con- bility towards the ligand. Quantitative binding studies were
A
B
-&
-
-
Ligand-bindingDomain of u-PAReceptor
7845
TABLE I NH2-terminal amino acid sequences of chymotryptic u-PAR fragments and partial sequences of intact u-PAR M, 16,000 fragment"," L R ? M Q ? ( K ) T N G D ? R V E E ? A ? G u-PAR residue 1-20' L R C M Q C K T N G D C R V E E C A L G
M,35,000-50,000 fragmenPd S R S R Y L E ?(I)S ? S R S R Y L E C I S C u-PAR residue 88-98' " Parentheses indicate that the identification was uncertain. Question mark indicates the lack of any identification. 'Determined by automatic sequencingof the electroblottedM,16,000 u-PAR fragment (band I of Fig. 2A ). The initial yield was 24 pmol of Leu at step1. The repetitive yield, based on Gly (steps 10 and 20) was 96%. No other sequence could be detected (sequencing limit2 pmol). Sequence obtained from NH2-terminal amino acid sequencing of the intact protein (17) and fromcDNA sequencing (18). Determined by automatic sequencing of the electroblotted M,35,000-50,000 u-PAR fragment (band I I of Fig. 2 A ) . The initial yield was 29 pmol of Ser at step 1. The repetitive yield, based on Ser (steps 1,3, and 10) was 90%. No other sequence could be detected (sequencing limit2 pmol; 3 pmol for Glu and Gly). Sequence deduced from cDNA sequencing(18).The preceding residue (i.e. no. 87) is a tyrosine. ~ ~ ~ 1 0 1 - 32
3
4
A ~ ~ ~ 1 01 - 3 2
FIG.3. Detergent phase separation ofu-PAR and its ligandbinding fragment. Purified u-PAR was degraded with chymotrypsin, asdescribed in the legend to Fig. 1. The sample was diluted100fold and subjected to temperature-induced phase separation in the presence of 1% Triton X-114. The resulting water phase(lune 3 ) ,the detergent phase (lane 2 ) , a sample of the degradation mixturewhich had not beensubjected to phaseseparation (lane I ) and a blind sample (i.e. chymotrypsin-treated buffer, not subjected to phase separation) ( l a n e 4 ) were incubated with '"I-ATF (1 nM) and subjected to chemical cross-linking with DSS. All samples were made up to the same final dilution factor (138-fold) during the assay by addition of a CHAPS-containing buffer. The cross-linked samples were analyzed by SDS-PAGE under reducing conditions followed by autoradiography of the gel. The electrophoretic mobilities of molecular mass marker proteins are indicated.
B ~ ~ ~ 1 0 1- 3
z
FIG. 4. Deglycosylationof the ligand-bindingfragment. Samples of intact, purified u-PAR (lanes AI and H I ) or chymotrypsin-treated u-PAR (lanes A2 and 8 2 ) were prepared as described in the legend t o Fig. 1, except that the chymotrypsin concentrationwas 200 ng/ml. The samples were diluted 67-fold, incubated with "Ilabeled ATF (1 nM) and subjected to chemical cross-linking with DSS. The cross-linked samples were pretreated for deglycosylation under denaturing conditions (see "Experimental Procedures") and incubated for 18 h a t 37 "C in the absence ( A ) or presence ( B ) of peptide:N-glycosidase F. The products were analyzed by SDS-PAGE under reducing conditions, followed by autoradiography. Theelectrophoretic mobilities of molecular mass marker proteins areindicated.
explained by the presence of N-linked carbohydrate. Thus, after deglycosylation, the conjugate of '2sI-ATF and the ligand-binding fragment migrated as anM,25,000 conjugate in not possible with the amountsavailable, but the activitywas good agreement with the expected size for an adduct formed demonstrated by chemical cross-linking after incubation with between ATF and an87-amino acid residue component. The the radiolabeled ligand present at 1 nM; i.e. using the same only potential N-glycosylation site within the region of amino conditions aspreviously found optimalfor the demonstration acid residues 1-87 of u-PAR is Asn-52 (18).Therefore, the of the binding activity of the intactreceptor (7, 17). present deglycosylation experiment identifies this asparagine Amino acidsequencing showed that the ligand-binding as a glycosylated residue. fragment had the same NH, terminus as uncleaved u-PAR, A further chymotrypticcleavage of the M,16,000 fragment and thatcleavage had occurred between Tyr-87 and Ser-88 of abolished the ligand-binding activity. This second cleavage the intact protein. The latter aminoacid residue constituted occurred rather close to one of the termini of the M, 16,000 the single detectable NH, terminus in the only other degra- polypeptide, since anM,13,000 product was demonstrated by dation product observed (i.e. the larger, non-ligand-binding SDS-PAGE under reducingconditions. However, it is not component), thusreflecting astrikingly specific cleavage. The known whether thecleavage site involved was situated in the ligand-binding fragmentthus probably covered the whole NH, or COOH terminus of the M,16,000 fragment, since the sequence 1-87, although additionalcleavages close to thenew amounts available were toosmall to allow NHz-terminal COOH-terminus cannot be excluded. The apparentmolecular amino acid sequencing of the M,13,000 product. All properties observed in the present study suggest that mass of this fragmentwhen analyzed by SDS-PAGE (i.e. M, 16,000) was somewhat higher than that expected for an 87- the NH,-terminal, M,16,000 fragment constitutes a distinct amino acid residue fragment, but this discrepancy could be structural and functionaldomain within u-PAR.Thus, itwas
Ligand-bindingDomain of u-PA Receptor
7846 u-PAR 1 u-PAR 2 U-PAR 3 co
LRmmKTN GDCRVE LECISSGSSD MSCERG RaYSGKGNS THGCSSE x x ~ x x ~ x x xgxxcxxx g
Ly-Ca Ly-6~
LEEYaYGVPFETSCPSI TEPYPDGVVQEAAV mYECYGVPIETSEPAV TCRASDGFCIAQNIEL IKCFVGNSYH QQNGDWFDNATHSVHXEPSQDRCRKIVQQI
sgp-2
co
u-PAR 1 U-PAR 2 U-PAR 3
co Ly-6a Ly-CC sgp-2 CQ
ECALGQDLC_RTTIVRLWEEGEELELVEKSCTHS
RHQSLQCRSPEEQCLDWTHWIQEGEEGRPKDDRHLRG ETFLINRGPMNQCLVATGTH EPKNQSYMVRG
gggggxCxxxxxxCxxxxxxxggggggxxxxxxxxxxx
IVDSQTRKVKNNL IEDSQRRKLKTRQ KLDEEWQVRYIRQ xx~xx~xxxxgxxx~xxxgggggggggx~xxxxxx~xxxxxxx XXDXXXXXXXXXX
EKTNRTLSYRT CGYLPGEPGSN CATASMQQHAHL XXXXXXxxxxxg
1
GLKITSLTEW QGLDUNQGNSGRAVTYSRSRY GFHNNDTFHFLKC GNTTKCNEGPILELENLPQNG GDAFSMNHIDVSC CTKSGENH PDLDVQYRS (anchor) ggxxxxxxxxxxx GxxxxCNxgxxxxxxxxx
-
anchor ? --- anchor ? anchor CxxxxxxxxxxgggggggxxxxxxxxTx~g~xxxx~N CLPIEPPNIESM
EILGTKVNVKTSE cQEDI.&N KDPNIRERTSC E S E D U N CAEGGEIGAYDGRVCKDRIGTSGVKMTYCHEQTEGCN
GLSFCPAGVPI
FIG. 5. Internal amino acid sequence repeats of u-PAR and homology of these repeats with the extracellular domains of T-cell-activating proteins/Ly-6 antigens and squid protein Sgp-2. Cysteine residues are underlined. Abbreviations are: u-PAR 1-3, the first, second, and third repeats of u-PAR (residues 192,93-191, and 192-282, respectively, of the amino acid sequence deduced from cDNA sequencing (18)); co, consensus sequence for the threerepeats of u-PAR Ly-Ga,Ly-Gc, Ly-6 antigens/T-cell activating proteins(residues 1-79 and 1-76, respectively) (28-31); Sgp-2,squid glycoprotein Sgp-2 (residues 1-92) (32); CO, consensus sequence for Ly-6a, LyGc, and Sgp-2. Anchor denotes the attachment sites for glycosyl-phosphatidylinositol tails. Question mark indicates that the attachmentsite was proposed by alignment with the Sgp-2 sequence (32). For u-PAR, the attachment site was tentatively assigned to either Ser-282 (i.e. the last residue shown) or to one of the residues Gly-283 or Ala-284 (36). In the consensus sequences shown, residues conserved in all sequences were defined as consensus residues, variable positions were marked with X and positions containing gaps in any of the member sequences were marked with g. The arrow indicates the bond cleaved during limited chymotryptic digestion. Note the similarity of the consensus sequence for u-PAR repeats with that for Ly-6 and Sgp-2 sequences (pattern of cysteines, location of gap regions).
a well-defined ligand-binding fragment, liberated by mild protease treatment, and itwas not disulfide-linked to therest of the protein (Fig. 1B). Furthermore, when subjected to Triton X-114 phase separation (ie. under non-denaturing conditions), itdissociated from the rest of the protein, and its lack of detergent binding suggests that it is water-soluble. Finally, its ability to bind the ligand was, at least qualitatively, independent on the rest of the protein, since its binding activity could be demonstrated after detergent phase separation in the same experiment. The experimental finding that the M, 16,000 fragment behaves as a distinct, structuraldomain is in accordance with an analysis of internal homology in u-PAR. This analysis reveals that theamino acid sequence of the receptor, deduced from cDNA sequencing (18), contains three repeats (repeat1, residues 1-92; repeat 2, residues 93-191; repeat 3, residues 191-282) characterized by a unique pattern of cysteine residues (Fig. 5). In this alignment, the sequences of the second and third repeats show 22% identity; all the 10 cysteines of the second repeat align with the 10 cysteines of the third repeat. The first repeat appears to be more distantly related (12 and 16% identity with thethirdand second repeats, respectively); 7 of its 8 cysteines align with those of the other two repeats. These observations imply that u-PAR is organized in threestructural domains; the functionally active fragment observed experimentally corresponds to the first repeat, liberated by cleavage of an inter-repeat bond (Fig. 5). This proposed domain-structure would thus also explain the striking specificity of chymotryptic digestion of u-PAR. The presence of the three repeats in u-PAR suggests that the receptor arose as a result of internal triplication of an ancestral domain. Indeed, a search of the protein sequence data base identified some homologous proteins possessing just a single copyof this cysteine-rich unit; the extracellular parts
of T-cell-activating proteins/Ly-6 antigens (28-31) and the Ly-6-related squid protein Sgp-2 (32) are related to the internal repeatsof u-PAR (Fig. 5). Interestingly, like u-PAR, these proteins are attached to the cell membrane by a glycosylphosphatidylinositol anchor (32,33). The existence of homologous glycosyl-phosphatidylinositol anchored proteins with a solitary copy of the repeat-type found in u-PAR underlines the structuralindependence of these domains. The functional importance which seems to be connected to u-PAR in plasmin-mediated cell surface proteolysis (13, 14) makes the present, separateligand-binding domain a valuable reagent asapotential soluble u-PAR antagonist.Thus,a soluble molecule whichcompetes with u-PAR for the binding of u-PA will be an importanttool for the study of cell surface plasminogen activation in cellular invasiveness (34, 35) and may also have a therapeutic potential for interference with these processes. Acknowledgments-The excellent technical assistance of Lene Askjer Sbrensen, Marianne Val10 Nielsen, Tore Sand@,and Linda Kirk Nielsen is gratefully acknowledged. Dr. G. Cassani is thanked for the generous gift of ATF. Note Added in Proof-After submitting the manuscript, we became aware that the human membrane inhibitor of reactive lysis (MIRL), CD59 (Sawada, R., Ohashi, K., Anaguchi, H., Okazaki, H., Hattori, M., Minato, N., and Naruto, M. (1990) DNA Cell Biol. 9, 213-220) is related toand is probably a human homologue of murine Ly-6 antigens. Sequence comparison has confirmed that CD59 is an additional member of the protein family with homology to theconsensus structure of the u-PAR repeats. REFERENCES 1. Dan@,K., Andreasen, P. A., Gdndahl-Hansen, J., Kristensen, P., Nielsen, L. S., and Skriver, L. (1985) Adu. Cancer Res. 44, 139-266 2. Saksela, 0. (1985) Biochirn. Biophys. Acta 823,35-65
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