A Novel Receptor Tyrosine Phosphatase-a That Is Highly Expressed ...

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May 5, 1993 - Olli Silvennoinen, and Joseph Schlessinger 11. From the ...... Fischer, E. H., Charbonneau, H. & Tonks, N. K. (1991) Science 253,401-. 3. Tonks ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 268, No. 33, Issue of November 25, pp. 24880-24886,1993 Printed in U.S.A.

Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc

A Novel Receptor Tyrosine Phosphatase-a ThatIs Highly Expressed in the Nervous System* (Received for publication, May 5, 1993, and in revised form, July 14, 1993)

Hai Yan, Albert Grossman, Hong Wang, Peter D’EustachioS, Kevin Mossiep, Jose M. Musacchioll, Olli Silvennoinen, and JosephSchlessinger 11 From the Departments of Pharmacology and $Biochemistry, New York University Medical Center, New York, New York 10016 and SSugen Inc., Redwood City, California 94063-4720

A novel transmembrane receptor protein tyrosine phosphatase-a (RPTP-a) was cloned from a rat brain stem cDNA library. The extracellular segment of one form of RPTP-a contains824 amino acids and iscomposed of three immunoglobulin-like and five fibronectin type I11 (FNII1)-like repeats. The 627-amino acid cytoplasmic region of RPTP-a consists of two catalytic domains oriented in tandem. Northern blot analyses indicate that RPTP-a is highly expressed in the brain 88 two major transcripts of 5.7 and 6.9 kilobases (kb). The 5.7-kb transcript is expressed exclusively in the brain while the 6.9-kb species can be detected in the lung and heart, but at significantly lower levels. In situ hybridization studies confirm that RPTP-u is localized predominately in the nervous system and can be detected in the rat as early as embryonic day 12. During embryonic development, RPTP-u isexpressed sysextensively in the central and peripheral nervous tems, including the trigeminal and dorsalroot ganglia as well as the retina. In adultrat brain, expression is restricted primarily to the olfactory tubercule, cerebellum, and hippocampus. Within the latter structure, RPTP-u is present in the pyramidal cell layer and granular layer of the dentate gyrus. Transfection of RPTP-a cDNA into human embryonic kidney 293 cells results in the synthesis of a protein with an apparent molecular mass of 200 kDa as detected by immunoprecipitation and immunoblot analyses using polyclonal antibodies against the FNIII-like repeats present in the extracellular domain of RPTP-a. The gene for RPTPa has been mapped to distal chromosome 17 in the mouse.

Protein tyrosine phosphorylation is critical for regulation of normal cell growth, proliferation, and differentiation. The extent of phosphorylation of tyrosine residues on cellular proteins is controlled by both protein tyrosinekinases (1)and protein tyrosine phosphatases (PTPs)’ (2). While a variety * This work was supported by Sugen Inc. (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll Recipient of Grant MH-17785. 11 To whom correspondence should be addressed: Dept. of Pharmacology, New York University, 550 First Ave., NewYork, NY 10016. Tel.: 212-263-7111; Fax: 212-263-7133. The abbreviations used are: PTP, protein tyrosine phosphatase; RPTP, receptor protein tyrosine phosphatase; FNIII, fibronectin type 111; LAR, leukocyte common antigen-related protein;N-CAM, neural cell adhesion molecule; PCR, polymerase chain reaction; bp, base pair; kb, kilobase.



protein tyrosine kinases have been identified and well characterized ( l ) , relatively little was known about protein tyrosine phosphatasesuntil tyrosine phosphatase 1B from human placenta was purified and subsequently cloned (3,4). To date, more than 26 PTPs have been identified ( 5 , 6 ) ,and theknown enzymes can be divided into two groups; a cytosolic form and a receptor-type form with a single transmembrane domain (RPTP). Most RPTPs contain two conserved catalytic tyrosine phosphatase (PTP) domains and each encompasses a segment of 240 amino acid residues ( 5 , 6).The catalytic domain contains a highly conserved motif of 11-amino acid residues, (I/V)HCXAGXXR(S/T)G, in which the cysteine residue was shown to be essential for protein tyrosine phosphatase activity (7-9). The RPTPscan be subclassified into five types based upon the amino acid sequence diversity of their extracellular segments (2, 5 , 6, 10, 11). The presence of both Ig-like and fibronectin type I11 (FNII1)-like repeats in the extracellular domain is characteristic of type I1 receptor tyrosine phosphatases, which includes LAR (12-14), PTPp (15), PTPK (16), and Drosophila DPTP (14). These structural features resemble the N-CAM family of cell adhesion molecules (17-20), suggesting type I1 receptor tyrosine phosphatases may function as cell adhesion molecules. Tyrosine phosphorylation initiated by several receptor tyrosine kinases has been implicated in neuronal cell proliferation and differentiation (21). These include the receptors for neurotrophic factors belonging to the tropomyosin-receptor kinase (trk) family of receptor tyrosine kinases, as well as platelet-derived growth factor and fibroblast growth factor receptor tyrosine kinases (22). Virtually nothing is known about the control of tyrosinephosphorylation by protein tyrosine phosphatases in the mammalian nervous system. The present report describes the cloning and characterization of a type I1 receptor tyrosinephosphatase, RPTP-a, which is highly expressed both in the developing and mature central and peripheral nervous systems of the rat. RPTP-a is a receptor protein having a single transmembrane, anextracellular segment consisting of three Ig-like and five FNIII-like repeats, and a cytoplasmic segment having two tandem catalytic domains. Transfection of RPTP-a cDNA into human embryonic kidney 293 cells results inthe synthesis of a protein with an apparent molecular mass of200 kDa. RPTP-u is a new member of the type I1 receptor tyrosine phosphatases (2, 5 ) that is most homologous to rat LAR (13), followed by human PTP-6 (23) and Drosophila LAR (14). The RPTP-a gene had been mapped to distal chromosome 17 of the mouse. EXPERIMENTALPROCEDURES

Materials-Chemical reagents were purchased from Sigma. The Xgtll rat brainstem cDNA library was obtained from Clontech.

24880

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brains Restriction endonucleases, modifying enzymes, terminal deoxynu- pH 7.4. Adult rats were sacrificed by decapitation, and then the cleotidyl transferase, ramdom priming kits, and Sephadex G-25 quick- were rapidly removed and frozen on dry ice. 20-pm sections obtained spin columns were purchased from Boehringer Mannheim. The with a cryostatmicrotome were postfixed for 30 min and thenwashed three times in 0.1 M sodium phosphate, pH 7.4. The sections were primers for PCR,RNA-PCR, and for sequencing, as well as the oligonucleotide probe for in situ hybridization, were synthesized by then dehydrated and stored a t -20 "C. Prehybridization and hybridization were carried out as described an automated DNA synthesizer (Applied Biosystems, model 380A). The latterwas purified by oligonucleotidepurification cartridge chro- elsewhere (11,26).Sections were mounted on the slides and incubated with 1 x IO6 cpm of labeled probe-1 in 10 mM dithiothreitol. The matography (Applied Biosystems). The PCR and RNA-PCRkits were purchased from Perkin-Elmer Cetus. Sequenase and random specificity of hybridization was determined by incubating an adjacent primer kits were obtained from U. S. Biochemical Corp. and RNA section with labeled probe-1 in the presence of a 70-fold excess of unlabeled probe-1. The slides were washed twice in 2 X SSC at room isolation kits from Stratagene. Isolation of Novel PTP Domains from Total RNAof PC12 Cells temperature (30 min), 1 X SSC at 50 "C (30 min), 0.5 X SSC at 50 "c by PCR-A pair of degenerate oligomers,5'-GA(T/C)TA(T/C)AT(T/ (30 min), and finally in 0.5 X SSC at room temperature (10 rnin). C)AA(T/C)GCI AG(T/C)TT-3' and 5'-A(T/C)ICCIGC(A/G)CT(A/ Sections were dehydrated and exposed to X-Omat film for 10 days. Transfection, Immunoprecipitation, and Immunoblot Analyses-A G)CA(A/G)TGIAC-3', corresponding to conserved amino acid stretches DYINAS and VCHSAG of known PTP domains, was used cDNA insert containing the entire open reading frame of RPTP-u in the RNA-PCR to specifically amplify novel sequences of PTP was subcloned intoan eukaryotic expression vector to generate domain from 1 pg of PC12 total RNA (following procedures recom- RKSigma and RKSigmaR, in which the cDNA insert was oriented in mended by Perkin-ElmerCetus).PCRproducts of expected size the sense or antisense direction, respectively. Human embryonic (about 450 bp) were isolated by agarose gel electrophoresis, purified, kidney 293 cells (27), grown to 20% confluence on fibronectin coated and then cloned into a pBluescript vector (Stratagene). ThecDNA dishes, were transiently transfected with appropriate plasmids by the inserts were sequenced by the dideoxy chain termination method calcium phosphate precipitation method (25). The transfected cells using the Sequenase Version 2.0 kit. Sequencing of about 100 individ- were harvested after 48 h and lysed in lysis buffer (137 mM NaC1, ual clones led to the identification of three PTP domains whose 10% glycerol, 1%Triton X-100, 0.5% deoxycholate, 0.1% SDS, 2 mM predicted sequences werehomologous to, but distinct from, corre- EDTA, 20mM Tris, pH 7.3) containing proteinase inhibitors (aprotinin andleupeptin (1pglml) and phenylmethylsulfonyl fluoride (100 sponding sequences of all known PTPs. Library Screening and Isolation of cDNA Clones-The cDNA insert pg/ml)). After centrifugation (15,000 X g, 15 min at 4 'C), the supernatants were analyzed by either first immunoprecipitated with FNIII a PTP domain, from one of the above PTP domains, referred to as the was amplified and labeled using a random priming labeling kit. The antibody followed by immunoblot analysis or directly by immunoblot labeled cDNA insert was then used as a probe to screen a rat brain analysis. The proteins resolved by SDS-polyacrylamide gel electrostem cDNA library (24). About 600,000 recombinants were screened, phoresis were transferred onto Nytran membrane, immunoprobed from which 17 positive clones were purified after the first round of with FNIII antibody, and visualized with 1251-proteinA afterexposure screening. The most 5' end of the longest cDNA insert isolated to x-ray film. Analysis of Tyrosine Phosphatase Actiuity-The synthetic peptide following the first round of screening was used in successive rounds of screening until overlapping cDNA clones that contained the entire poly-Glu-Tyr was phosphorylated by treatment with purified epidercoding sequence for RPTP-a were obtained. Both strands of several mal growth factor receptor kinase domain in the presence of [y-"P] overlapping cDNA clones were sequenced using a Pharmacia DNA ATP andMn2+.The lysates from either untransfected or transfected human 293 cells were immunoprecipitated with anti-RPTP-a FNIII sequenator. Generation of Antibodies against Glutathidne S-Transferase Fusion antibody (see above). The immunocomplex was suspended in 50 p1 of buffer P (25 mM Hepes, pH 7.0,5 mM EDTA, 10 mM P-mercaptoethProtein-AcDNA fragment coding for amino acids 512-835of anol) and incubated with phosphorylated substrate at 37 "C for 30 RPTP-a (corresponding totheFNIII region of the extracellular min with shaking (28). Reactions were terminated by addition of an domain) was amplified by PCR and subcloned into a pGEXbacterial expression vector (Pharmacia LKB Biotechnology Inc.) to construct acidic charcoal mixture. PTP activity was monitored by following the plasmid pGEX-FNIII. This plasmid was transformed into Escherichia appearance of free 32Pin the reaction supernatants. incoli strain DH5a. After isopropyl-1-thio-8-D-galactopyranoside Mapping the Mouse RPTP-u Gene (Table I)-Inbred and recomduction, the insoluble glutathione S-transferase fusion protein was binant inbred (RI) mice were obtained from The Jackson Laboratory isolated by SDS-polyacrylamide gel electrophoresis. Two rabbits were (Bar Harbor, ME). Genomic DNA was isolated from liver, digested immunized with the fusion protein to produce polyclonal antisera with restriction enzyme TaqI, and thenanalyzed by Southern blotting (FNIII Ab) against RPTP-a. with probe pGEX-FNIII (see above) as described previously (29). To RNA Isolation and Northern Blot Analysis-Total RNA was iso- establish genetic linkages, the distribution patterns observed for lated from different tissues of 6-day-old rats using RNA isolation kits RPTP-a in the RI strains were compared to approximately 1360 other obtained from Stratagene. Samples containing 30 pg of total RNA markers maintained in a database a t New York University? Signifiwere resolved in a formaldehyde/agarose-gel(24)and thentransferred cance of matches was assessed using the Bayloc algorithm (30). to Nytran membrane (Schleicher & Schuell). Probes corresponding to different cDNA regions of RPTP-u were amplified by PCR, puriRESULTS fied, and labeled using a random priming kit. Hybridization and subsequent washing were carried out essentially as described elseMolecular Cloning of the Full-length cDNA for RPTP-awhere (25) with an additional wash carried out in 0.1 X SSC and Three cDNA fragments coding for novel PTP domains 0.1% SDS at60 "C for 15 min. were initially identified in the total RNA isolated from PC12 In Situ Hybridyzation-Probe-1, 5"TAGACCACAATGGAACCATCGTTGTCAGGCTTTGGGGCGACACTAGGCTT-3', was syn- cells by reverse PCR with the primers for the conserved thesized in an Applied Biosystems 380A DNA synthesizer and then regions of other known PTPs. One of these PTP domains, purified. Probe-1 is antisense and complementary to the cDNA of termed the u PTP domain, was expressed at high levels in RPTP-a from nucleotide 2918-2967; a region that encodes amino the brain and only to a limited extent in the lung and intestine. This probe was chosen Based on these observations, a random and oligo(dT)-primed acid stretch 694KPSVAPKPDNDGSIVVY710. because it is least homologous to the corresponding sequences in rat rat brain stem cDNA library (Clontech Inc.) was screened for LAR and human PTP-6. Probe-1 was labeled a t 3'-end with dATP (using a terminal deoxynucleotidyl transferase kit) and then the full-length cDNA of RPTP-a using the PCR fragment purified by Sephadex G-25 column chromatography. The specific initially cloned from the RNA of PC12cells. After three activities of the labeled probe ranged from 1 to 4 X IO8 cpmlpg of rounds of screening, several overlapping clones were isolated. DNA. They were sequenced in both directions. As shown in Fig. 1, Adult male and timed-pregnant female Spragne-Dawley rats and the 5.7-kb full-length cDNA for RPTP-a was constructed by their litters were used for the study. The day of successful copulation was determined by the presence of sperm in vaginal smears and was joining the two longest overlapping clones (H-29 and B-10) recorded as embryonic day 0 (EO). The day of birth was considered at a unique MluI site. The 5.7-kb cDNA has an open reading postnatal day 0 (PO).Whole bodies of E12 embryos were fixed in 4% frame of 1501 amino acid residues starting from the ATG at paraformaldehyde in 0.1 M sodium phosphate, pH 7.4, for 4 h followed by overnight infiltration with 15% sucrose in 0.1 M sodium phosphate, P. D'Eustachio, unpublished results.

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FIG. 1. Restriction map of cDNA clone encoding rat RPTP-a. The thin lines indicate several overlappingcDNA clones; both strands of each clone were sequenced. Unfilled area represents the open reading frame for RPTP-o containing the putative signal peptide (S) and transmembrane domain (TM). The thick lines indicate the 5’-and 3’-untranslated sequences. Restrictionsites are: E, EcoRI; B, BamHI; BL, BglII; ML,MluI; S , StuI; A P , Asp700; K , KpnI; SP, SplI). A scale bar in kilobases (kb) is at the top.

bp 839. The ATG was preceded by stop codons in all three identity with PTP domain I1 (Fig. 2 A ) . Thecysteine residues, which have been implicated to be essential for phosreading frames and meets the requirements for the translation phatase activity, are conserved in both PTP domains. The initiation site (31). There is an 838-bp 5”untranslated sequence and a 353-bp 3”untranslated sequence containing a overall cytoplasmic segment of RPTP-a has a striking sequence homology to that of rat LAR(84% identity)and poly(A) tail. of Characterization of RPTP-a-The deduced amino acid se- human PTP-6 (86% identity). Furthermore, comparison quenceforRPTP-aisshownin Fig. 2A. Itcontains 23 the cytoplasmic segmentsof RPTP-a and ratLAR indicates hydrophobic amino acidslikely to function asa signal peptide that a higher degree of identity is presentbetween domain I1 (32), followed by an 824-aminoacid extracellular domain and (93%) than domain I (84%). A schematic diagram showing the multiple domain structure of RPTP-a is presented in Fig. a 24-hydrophobic amino acids transmembrane domain. The 627-amino acid cytoplasmic region contains two tandem PTP 20. Although RPTP-a is highly homologous to rat LAR, espedomains. The overall sequence of RPTP-a demonstrates a high degree of similarity to rat LAR (13), having cially in the cytoplasmic region, the diversity of their extra71% sequence identity within the entire coding region. RPTP- cellular domains, particularly in the FNIII-type repeats, demis member of the typeI1 receptor a is also 75% identical t o partially sequenced human PTP-6 onstrates that RPTP-aa new tyrosine phosphatases. (23). Distribution of RPTP-a mRNA-Northern blot analyses of The extracellular segmentof RPTP-a consists of three Ig6-day-old rat tissues are like domains (residue 33-315) (33) and 5 FNIII-like repeats total RNA isolated from various presented in Fig. 3A. The blot depicted in this figure was (residue 320-786) (34, 35). Search in database revealed sigprobed with a cDNA fragment coding for amino acid residues nificant homology to the extracellular segments of rat LAR 408-521 of RPTP-a. Identical results were obtained with a (63% identity). Also, the homology between the Ig-like docDNA probe specific for a cytoplasmic segment of RPTP-a mains of RPTP-a and LAR (75% identity) is greater than and with oligonucleotide probe-1 (data not shown). RPTP-a that of theFNIII-likerepeats (54% identity).There was is highly expressed in the brain as compared to other tissues. significant homology also between the FNIII-like repeats of Of the two major RPTP-a transcripts(5.7 and 6.9 kb) expresRPTP-a and human PTP-8. In addition, the extracellular sion of the 5.7-kb speciesis detected exclusively in brainwhile sequence of RPTP-a is homologous to neural cell adhesion the 6.9-kb transcript, presentedalso in the brain, is expressed molecules in terms of their overall structure and sequence. to a significantly lower level in lung and heart. Upon longer The highest homology was detected with L1 (26% identity) exposure, the 6.9-kb species can also be detected in kidney (36), followed by N-CAM (22% identity) (18) and neuroglian and intestine. The cDNA that wassequenced and described (22%) (37). Fig. 2B shows alignment of three Ig domains of in this paper probably corresponded to the 5.7-kb mRNA.3 of rat LAR The nature of these various mRNA species is not clear. Fig. RPTP-a toeach other and to the first Ig domain and mouse N-CAM, respectively. Within each Ig domain, two 3B shows a short exposure of same Northern blot indicating cysteines are thought t o be involved in intradomain disulfide RPTP-a is highly expressed in the brain. Fig. 3C is the gel binding and areconserved (Fig. 2B, asterisks). The alignment stained with ethidium bromide revealingthe integrityof RNA. of the five FNIII-like repeatsof RPTP-a to each other and to In Situ Hybridization-Since RPTP-a is highly expressed the first FNIII-like repeat of rat LAR and type I11 repeat in the brain as detected by Northern blot analysis, its expres(domain 7) of human fibronectin is shown in Fig. 2C. The sion at specific sites withinwhole embryos and adult rat brain fifth repeat of RPTP-a exhibits greater divergence from con- was then identified by in situ hybridization. Using labeled sensus than the other four. Also, the extracellular region of probe-1, an antisense oligomer, the expression of RPTP-a RPTP-a does not contain the RGDsequence that is required was detected in brain, spinal cord, and dorsal rootganglia as for fibronectin binding (38). early as embryonic day E12 (Fig. 4A).No apparentexpression The cytoplasmic segment of RPTP-a, like most receptor was detectedinother tissues. Hybridization signals were tyrosinephosphatases,contains two conserved PTP doB. Goldstein, personal communication. PTP domain I shows 47% sequence mainsintandem.

Novel Receptor Tyrosine Phosphatase A. 1

MAPTWRPSW SWGPVGLFL VLLARGCLAE EPPRFIREPK DQIGVSGGVA

51

SFVCQATGDP KPRVTWNKKG KKVNSQRFET IDFDESSGAV LRIQPLRTPR

101

DENWECVAQ NSVGEITVHA KLTVLREWL PPGFPNIDHG PQLKWERTR

151

TATMLCAASG NPDPEITWFK DFLPVDPSAS NGRIKQLRSG ALQIESSEET

201

DQGKYECVAT NSAGVRYSSP ANLYVRVRRV APRFSILPMS HEIMPGGNVN

251

ITCVAVGSPM PYVKWMCGAE DLTPEDDMPV GRNVLELTDV KDSANYTCVA

301

MSSLGVIEAV AQITVKSLPK APGTPVVTEN TATSITVTWD SGNPDWSYY

351

VIEYKSKSQD GPYQIKEDIT TTRYSIGGLS PNSEYEIWVS AVNSIGQGPP

401

SESWTRTGE QAPASAPRNV QARMLSATTM IVQWEEPVEP NGLIRGYRVY

451

YTMEPEHPVG NWQKHNVDDS LLTTVGSLLE DETYTVRVLA FTSVGDGPLS

501

DPIQVKTQQG VPGQPMNLRA EAKSETSIGL SWSAPRQESV IKYELLFREG

551

DRGREVGRTF DPTTAFWED LKPNTEYAFR LAARSPWLG AFTAWRQRT

601

LQAISPKNFK VKMIMKTSVL LSWEFPDNYN SPTPYKIQYN GLTLDVDGRT

651

TKKLITHLKP HTFYNFVLTN RGSSLGGLQQ TVTARTAFNM LSGKPSVAPK

701

P D N D G S I W LPDGQSPVTV QNYFIVMVPL RKSRGGQFPI LLGSPEDMDL

751

EELIQDLSRL QRRSLRHSRQ LEVPRPYIAA RFSILPAVFH PGNQKQYGGF

801

DNRGLEPGHR WLFVLAVLQ KNEPTFAASP FSDPFQLDNP DPQPIVDGEE

851

GLIWVIGPVL AVVFIICIVI AILLYKNKPD SKRKDSEPRT KCLLNNADLA

901

PHHPKDPVEM RRINFQTPGM LSHPPIPITD MAEHMERLKA NDSLKLSQEY

1251 1301 1351 1401

1451 1501

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LPCNKF KNRLVNILPY ESSRVCLQPI RGVEGSDYIN ASFIDGYRQQ

KAYIATCGPL AETTEDFWPA LWENNSTIW MLTKLREMGR EKCHQYWPAE

RSARYQYFW DPMAEYNMPQ YILREFKVTD RRDGQSRTVR QFQFTDWPEQ GAPKSGEGFI DFIGQVHKTK EQFGQDGPIS VHCSAGVGRT GVFITLSIVL

ERMRYEGWD IFQTVKVLRT QRPAMVQTED EYQFCFQAAL EYLGSFDHYA

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Slgrna12!

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FIG. 2. Amino acid sequence and schematic diagram of rat RPTP-o. A , the amino acid sequence of RPTP-a deducedfrom overlapping cDNA clones is shown in one-letter code.The predicted signal peptide and transmembrane regions are underlined and two tandem tyrosine phosphatase domains are boxed. B , proposed alignment of three Ig-like domains of RPTP-u to the first Ig-like domain of rat LAR (13) and mouse N-CAM (18).The residue numbers of the initial and end amino acids of each repeat are indicated at the left and right. Conserved residues in these repeats are highlighted in bold letters. The cysteine residues are indicated by asterisks. C,proposed alignment of five fibronectin type I11 repeats of RPTP-a to the first FNIII-like repeat of rat LAR (13) and to the human fibronectin type 111 repeat of domain 7 (34). The residue numbers of the initial and end amino acids of each repeat are indicated at the left and right (except for human FNIII-7). Conserved residues in these repeats are highlighted in bold letters. D,schematic representation of PTP-a sequence showing the possible different functional domains.

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specific since theywere completely chased by a 70-fold excess of unlabeled probe-1 (Fig. 4B). In addition, a relatively low level of the expressionof RPTP-a was detected in the lung at embryonic day18 (EM), and the intensity of RPTP-a expression overall gradually decreased during embryonic development (data not shown). In the adult rat brain, expression of RPTP-u was confined to specific regions of the brain that included the olfactory bulb, cerebellumand hippocampus (Fig. 4C). Within the latter structure, the pyramidal cell layer and - 4.4 granular layer of dentate gyrus were labeled specifically (Fig. 4, C and D). Transient Expression of RPTP-a-Human embryonic kidney 293 cells (27), commonly used for overexpressing exoge- 2.4 nous proteins,were transfected transiently with a mammalian expression vector that directs the synthesis of RPTP-a in - 1.4 order to investigate its biochemicalproperties. The transfected cells were lysed in lysis buffer, and the supernatants were then subjected to immunoprecipitation with polyclonal FNIII antibody. This was followed by immunoblot analysis -0.24usingthesameantibodies.Theresultsshownin Fig. 5A revealed that FNIII antibody can specifically detect a protein of 200 kDa in supernatants from cells transfected witha C plasmid containing RPTP-a cDNA (RKSigma) (lane 1 ) but not from those transfected with a plasmid containing control DNA (RKSigmaR) (lane 2 ) . No band corresponding to the 200-kDa protein was detected in control immunoprecipitation experiments using preimmune serum (data not shown). The apparent molecular mass of the expressed protein after SDSpolyacrylamide gel electrophoresis is ingood agreement with the expected size for RPTP-a. The bands detected in lane 2 of Fig. 5, A and B, were nonspecific since they were also observed after incubation with preimmune serum. Several members of the typeI1 receptor tyrosine phosphatases, such as LAR (13, 40) and RPTP-K (16),undergo proFIG.3. Northern blot analysis of PTP-a expression in post- teolytic processing after synthesis. T o investigate whether natal rat tissues. 30 of total RNA prepared from 6-day-old rat similar processing occurs with RPTP-a, a new member of this tissues, as indicated, were used for Northern blot analysis. The blot group, the supernatants of lysates from the transfected cells was hybridized with a cDNA probe encoding aminoacid residue 408were subjected directly to immunoblot analysis using FNIII 521 of the extracellular domain of RPTP-a. Thesizes of RNA markers (Life Technologies, Inc.) are in kilobases (kb). A , gel exposed for 18 antibody or preimmune serum. The results presented in Fig. 5B show that in addition to the 200-kDa protein previously h; B, for 3 h. C, RNA gel stained with ethidiumbromide. identified, an additional protein with an apparent molecular mass of 100 kDa was detected in lysates of cells transfected with plasmid RKSigma (lane 1), but not with RKSigmaR (hne2 ) . No such bands a t 200 and 100 kDa were detected with preimmune serum. Identical results also were obtained

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69

2

FIG. 4. I n situ hybridization analysis of RPTP-a expression in rat. A , sagittal section of whole rat at embryonic day 12 (E12) was hybridized with labeledprobe-1 (see “Experimental Procedures”). FIG.5. Transient expression of RPTP-u in human embrycx, cortical neuroepithelium; 4V, fourth ventricle; LV, lateral ventri- onic kidney 293 cells. A , immunoprecipitation andimmunoblot of transientlytransfected withRKSigma or cle; SC, spinal cord; DRG, dorsal root ganglia. C, sagittal section of RPTP-o. Cellswere adult rat brain was hybridized with labeled probe-1. Cb, cerebellum; RKSigmaR. Supernatants of lysates were immunoprecipitated with Py,pyramidal cell layer; GrDG, granular layer of dentate gyrus; OB, polyclonal FNIII antibody and then immunoblottedwith same antiolfactory bulb. B and D, sections adjacent to A and C, respectively, bodies. Lane 1, lysate from cells transfected with RKSigma; lane 2, B, immunoblot analysis werehybridizedwithlabeled probe-1 in the presence of a 70-fold lysate fromcells transfected with RKSigmaR. excess of unlabeled oligomer to demonstrate thespecificity of in situ of RPTP-u with polyclonal FNIII antibody. Lanes 1 and 2 are same hybridization. as in A . Molecular mass markers areshown in kilodaltons.

Phosphatase Tyrosine Receptor Novel in immunoblot analyses of supernatants from lysates of transfected COS-1 cells (data not shown). These results suggest that RPTP-o also undergoes proteolytic processing and the 100-kDa protein may represent a cleavage product. PTP Actiuity of RPTP-o-To demonstrate that RPTPo has the intrinsic tyrosine phosphatase activity,lysates from human 293 cells transfected with RKSigma or RKSigmaR, respectively, were incubated with anti RPTP-a antibodies. The immunocomplexes were incubated with radiolabeled peptide. The tyrosine phosphatase activity of RPTP-o was assayed by measuring the amount of free 32Preleased. As shown in Fig. 6, approximately 7-fold increase in tyrosine phosphatase activity was observed in the immunocomplex from cells transfected with RKSigma as compared to those with RKSigmaR. Mapping the Mouse RPTP-a Gene-When TaqI-digested genomic DNA from various inbred strains was analyzed by Southern blotting using an RPTP-u probe (pGEX-FNIII), two allelic forms of the gene were detected (Table IA). The pattern of inheritance of these alleles in recombinant inbred strains of mice defined a single genetic locus (TableIB). Comparison of the inheritance pattern for this locus with these previously established for approximately 1360 markers distributed over the entire mouse genome allowed us to map the locus for RPTP-o todistal mouse chromosome 17.

24885 TABLEI Use of DNA restriction fragment length variants to map RPTP-u to mouse chromosome 17 (A) Variant DNA fragments (sizes in kb) in Southern blots of TuqI-digested genomic DNA. (B) All RI strains were homozygousfor one of the progenitor strain alleles of RPTP-u, as indicated by the generic symbols 9, 129/J-like, A, AKR/J-like; B, C57BL/GJ-like; C, BALB/c-like; J , SJL-like; L, C57L/J-like; N, NZB/BlNJ-like; and S, STS/A-like. (C) The strain distribution pattern for RPTP-u was compared to ones for 436 other markers. All concordances whose odds of chance occurrence are less than 0.05 are shown. For each such match, the marker’s name and chromosomal location are shown, together with the observed recombination fraction (R/N), odds of observing that fraction or a smaller one by chance (30), and, conditional on the existence of linkage, the estimated distance in cM between the two loci (53), and the 95% binomial confidence limits for that estimate (39). A. Alleles of RPTP-o defined by Southern blotting

allele sire

strains

a

5.6

b

5.4

AKX,O2o/A,DBA/2J,C57BL/6J,C3H/Hel, BALB/c,NZB/BINl.SM/J.A/J,PL/J C57L/J,SWR/J,SJL/~,STS/A,l29/J

B. Inheritance of R F T P a alleles in R I strains

AKXL 1111112222233

-

567892346791458978um

LAAAA LAALLAALLL LLL JEi

cxs

Itl RK SigmaR

1

CSSSS SCCCC CSCS CCJJCCC

We report, presently, the molecular cloning, chromosomal localization, and expression profile of RPTP-o. A data base search revealed that RPTP-a is structurally similar to rat LAR (13) with an overall sequence identity of 71%. Similarity among the cytoplasmic domains (84%) is greater than thatof the extracellular domains (63%). RPTP-a was mapped to distal chromosome 17 of the mouse, which corresponds in part to human chromosomes 6 and 19. Hence, we conclude that RPTP-o is a novel member of the type I1 receptor tyrosine phosphatases (2). RPTP-o is highly expressed in the brainas two major transcripts (5.7 and 6.9 kb). Northern blot analysis and in situ hybridyzation demonstrated that the 5.7-kb transcript is expressed exclusively in thenervous system. The 6.9-kb transcript can be detected in lung, heart, kidney, and intestine, but at significantly lower levelsthan thatfound in brain. The

No DNA

N9N

CXJ

11111

DISCUSSION

BXJ NX129 11

I RK

Sigma

FIG. 6. Tyrosine phosphatase activity of RPTP-o. Lysates from human 293 cells eitheruntransfected or transfected with RKSigma or RKSigmaR, as indicated, were incubated with antibodies against RPTP-u. The immunocomplexes were incubated with radiolabeled peptide. The tyrosine phosphatase activity of RPTP-u was expressed as percentage of 32Preleased from total radiolabel input. The results are presented as the mean of five determinations f S.D.

C. Linkage relationships deduced from RI typingdata WN Odds Distance (95% lim) Marker Chr H-2 17 8/39 0.03248 7.4 (2.7-20.1) C3 17 0/14 0.W634 -.(43.9) Ras12-317 0/18 O.OW49 -.(~5.4) Hprt-2ps 17 1/18 0.00972 1.5 (0.0-11.6)

5.7- and 6.9-kb transcripts aremost likely generated by alternative splicing of the RPTP-o gene.3 Interestingly, human PTP-P was restricted to the developing central nervous system (26) while RPTP-o was readily detected in boththe peripheral and central nervous systems in the embryonic and adult rat. This suggests that RPTP-a plays a broad role in development and maintenance of the nervous system. The major difference between RPTP-o and LAR, which share ahigh degree of sequence homology, is their expression pattern in various tissues. LAR has abroad tissue distribution, being detected in a number of epithelia as well as in smooth and cardiac muscle (40). In contrast, the 5.7-kb transcript of RPTP-U, themost abundant species, is expressed exclusively in nervous tissues. Also, DLAR (14),the proposed counterpart of LAR in Drosophila, was found to be expressed predominately in the axons in thecentral nervous system of Drosophila embryos (41). Unlike rat LAR whichhas 6predicted N-glycosylation sites, no such sites have been found in the extracellular domain of RPTP-o. However, this domain is rich in serine and threonine residues (16%) andlikely to provide multiple attachment sites for 0-linked glycosylation. Moreover, RPTP-o has only five FNIII repeats while LAR contains eight. It is possible that larger alternatively spliced transcript (6.9 kb) encodes a protein containing eight FNIII-likerepeats? Like other type I1 receptor tyrosine phosphatases, such as PTP-p, RPTP-K,Drosophila LAR, and Drosophila PTP (14), the extracellular segment of RPTP-a consists of both Ig- and FNIII-like repeats. This structuralfeature also has been identified in neural cell adhesion molecules such as N-CAM, neuron-glia (Ng-CAM), and Ng-CAM-related (Nr-CAM) (19, 20). It has been demonstrated that the extracellular domain, especially the Ig-like repeats of these neural adhesion mole-

Novel Receptor Tyrosine Phosphatase

24886

10. Krueger, N. K. & Saito, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,7417cules, is crucialfor mediating homophilic and heterophilic 7421 binding when expressed in transfected cells (42, 43). Since 11. Barnea, G., Silvennoinen, 0..Shaanan, B., Honnerger, A. M., Canoll, P. D., D'Eustachio, P., Morse, B., Levy, J. B., Laforgia, S., Huebner K., there is significant homology among the extracellular domains Sap, J., Musacchio, J. M. & Schlessinger, J. (1993) Mol. Cell. BioL'l3, 1497-1506 of RPTP-a and L1, N-CAM and neuroglial (26,22, and 22% 12. Streuli, M., Krueger, N. K., Hall, L. R., Schlossman, S. F. & Saito, H. identity, respectively), itis possible thattheextracellular (1988) J. Ex Med. 1 6 8 , 1523-1530 which, in turn, 13. Yu, Q.,Lena&, T. & Wemberg, R. A. (1992) Oncogene 7 , 1051-1057 domain of RPTP-u also mediate such binding 14. Streuli, M., Krueger, N. K., Tsai, A. Y. M. & Saito, H. (1989) Proc. Natl. couples the tyrosine phosphatase activity to some intracellular Acad. Sei. U. S. A. 86,8698-8702 15. Gebbink, M. F. B. G., van Etten, I., Hateboer, G., Suijkerbuijk, R. Bei'erssignaling pathway. bergen, R. L., van Kessel, A. G. & Moolenaar, W. (1991) FEES t t t . Immunoblot analyses suggest that RPTP-a, like LAR and 290,123-130 Jiang, Y.-P. Wang, H., D'Eustachio, P., Musacchio, J. M., Schlessinger, J. 16. RPTP-K,undergoes proteolytic processing. Sequence analysis & Sap, J. (1993) Mol. Cell. Blol. 1 3 , 2942-2951 indicates thatRPTP-a has several potentialcleavage motifs, 17. Cunningham, B. A., Hemperly, J. J., Murray, B. A,, Prediger, E. A., & Edelman, G . M. (1987) Science 2 3 6 , 799-806 Brackenbu R. such as RK at residues 731-732, RR at 762-763, and RHSR 18. Barthels, D., Tintoni, M.-C., Wille, W., Ruppert, C., Chaix, J.-C., Hirsch, M.-R., Fontecilla-Camps, J. C. & Goridis, C. (1987) EMBO J. 6 , 907a t residues 766-769; all located within the fifth FNIII repeat 914 of the extracellular domain. These sitesmay serve as specific 19. Edelman, G. M. & Crossin, K. L. (1991) Annu. Rev. Bioehem. 6 0 , 155-190 20. Grurnet, M. (1991) Curr. Opin. Neurobiol. 1,370-376 cleavage signals for proteolytic processing (44). 21 Chno. M. ~,_ _ V.. (1992) "", Cell RR. ", 995-997 ._ Numerous studies have indicated that CD45 plays a crucial 22. Chao, M. V. (1992) Neuron 9 , 583-593 Krueger, H. X., Streuli, M. & Saito, H. (1990) EMBOJ. 9, 3241-3252 role in coupling the T-cell antigen receptor to a intracellular 23. 24. Sambrook. J. Fritsch. E. F. and Maniatis. T.(1989) Molecular Cloning: A signalingpathway (45-47). Experiments have suggestedCD45 Laboratory Manod, 2nd Ed., Cold SpringHarborLaboratory, Cold Spring Harbor, NY can activateIck or fYn protein tyrosine kinasesby dephospho- 25. Ausubel F. M., Brent R Kinston, R. E. Moore, D.D., Seidman, J. G., Smiti. J. A. & Struh. K . (1989) Current Protocols in Molecular Biologv.. rylation at inhibiting sites at the carboxyl terminus (48, 49). Wiley'hterscience, NY In the mammalian nervous system several tyrosine kinases 26. Levy, J. B., Canoll, P. D., Silvennoinen, 0.. Barnea, G., Morse, B., Honnerger, A. M., Huang, J-T., Cannizzaro, L.A., Park, S-H., Druck, T., such as c-yes (50),and the neuron-specific form of c-src (51) Huebner. K.. SaD. J.. Ehrlich. M.. Musacchio. J. M. & SchlessinEer. _ .J. have been shown to be expressed at high levels in the brain. (1993) J.'BiOl. Chlm.'268, 10573-'10581 27. Graham, F. L. & Smiley, J. (1977) J. Gen Virol. 36.59-74 Moreover, protein tyrosine kinase inhibitors have been shown 28. Tsai, A. Y., Itoh, M., Streull, M., Thal, T. and Saito, H. (1991) J. Biol. (52). to block long term potentiation in the hippocampus Chem. 2 6 6 , 10534-10543 P., Jadidi, S., Fuhlhrigge, R. C., Gray, P. W., and Chaplin, D. Furthermore, it has been demonstrated that several receptor 29. D'Eustachio, D. (1987) Kmmuno enetics 26,339-343 tyrosine kinases, such as nerve growth factor and fibroblast 30. Blank, R. D., Campfell, G. R., Calabro, A,, and D'Eustachio, P. (1988) Genetics 1 2 0 . 1073-1083 growth factor receptors (22), play a central role in neural 31. Kozak, M . (1984) NucleicAcid Res. 12,857-871 32. von Heijne, G. (1986) Nucleic Acid Res. 14,4683-4691 development. The molecular cloning of RPTP-u, which is 33. Williams, A. F. (1987) Krnrnunul. Today 8,298-303 highly expressed in both the central and peripheral nervous 34. Kornblihtt. A. R.. Umezawa. K.. Vibe-Pedersen. K. & Baralle. F. (1985) EMBO~J. 4,1755-1759 systems of embryo and adults,will enable us to design exper- 35. Hynes, R. (1985) Annu. Reu. Cell E d . 1,67-90 iments for determining the role of tyrosine phosphatases in 36. Moos, M., Tacke, R., Scherer, H., Teplow, D., Fruh, K. & Schachner, M. (1988)Nature 334,701-703 mammalian neurogenesis and maintenance. 37. Bieher, A. J., Snow, P. M., Hortsch, M., Patel, N. H., Jacobs, J. R., " "

I

"

~

"

'

'

Acknowledgments-We thank Moosa Mohammadi for labeling the tyrosine phosphatase substrate, Kiki Nelson for synthesizing primers and peptides, and Virginia Clarke for excellent technical help. We acknowledge Drs. Jan Sap, Ed Skolnik, Danila Rotin, Gilad Barnea, Martin Grumet, Barry Goldstein, Jiaoti Huang, Qiang Yu, and Peter D. Canoll for their helpful discussions. REFERENCES 1. Schlessinger, J. & Ullrich, A. (1992) Neuron 9 , 383-391 2. Fischer, E. H., Charbonneau, H. & Tonks, N.K. (1991) Science 253,401406 3. Tonks, N.K. Diltz, C. D. & Fischer, E. H. (1988) J. Biol. Chem. 263,67226730 4. Tonks, N. K. Diltz, C. D. & Fischer, E. H. (1988) J . Biol. Chem. 263,67316737 5. Saito, H. & Streuli, M (1991) Cell Growth & Differ. 2 , 59-65 6. Charbonneau, H. & Tonks, N. K. (1992) Annu. Reu. Cell Biol. 8,463-493 7. Streuli. M.. Krueeer, N. K.. Tsai, T.,. Tang, - M. & Saito, H. (1990) EMBO J. 9,. 2399-2407 8. Pot, D. A. & Dixon, J. E. (1992) J. Biol. Chern. 2 6 7 , 140-143 9. Wang, Y. & Pallen, C. J. (1991) EMBO J. 10,3231-3237

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