Murine Interleukin 2 Receptor J8 Chain

Home , CD4

Proc. Natl. Acad. Sci. USA Vol. 87, pp. 1806-1810, March 1990 Immunology Murine interleukin 2 receptor j8 chain: Dysregulated gene expression in lymphoma line EL-4 caused by a promoter insertion TAKESHI KONO*, TAKESHI DoI*, GEN YAMADA*, MASANORI HATAKEYAMA*, SEIJIRO MINAMOTO*, MITSURU TSUDOt, MASAYUKI MIYASAKAt, TAKASHI MIYATAt, AND TADATSUGU TANIGUCHI* *Institute for Molecular and Cellular Biology, Osaka University, Yamadaoka 1-3, Suita-shi, Osaka 565, Japan; tThe Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113, Japan; and *Department of Biology, Kyushu University Faculty of Science, Higashi-ku, Fukuoka 812, Japan Communicated by Charles Weissmann, December 4, 1989

ABSTRACT The functional, high-affinity interleukin 2 IL-2RP gene by the insertion of an intracisternal A particle receptor (IL-2R) consists of at least two receptor components, (IAP) element. IL-2Ra (p55) and IL-2Rf (p70-75). The cDNA encoding the murine IL-2Rfi has been isolated by using the previously cloned MATERIALS AND METHODS cDNA for human IL-2R8 as a probe. Analysis of the cDNA revealed that the murine IL-2RB shows a marked homology cDNA Cloning. To prepare mouse cDNA libraries, cDNAs with the human IL-2RB and that it is also structurally related were prepared by using poly(A)+ RNAs isolated either from to other cytokine receptors such as erythropoietin receptor. Con A-stimulated spleen cells of C57BL/6 mice or from The cDNA-directed murine IL-2Ri formed high-affinity IL-2R benzo[a]pyrene-induced lymphoma line EL-4 (C57BL/6 or- in conjunction with the endogenous IL-2Ra in a murine igin) (15), according to standard procedures (16). The spleen pro-B-cell line and could transduce IL-2-induced growth sig- cell-derived and EL-4-derived cDNAs were cloned into AgtlO nal. In mouse lymphoma line EL-4, the IL-2RB gene was found and CDM8 vectors, respectively, as described (16, 17). The the insertion the terminal repeat AgtlO library was screened for the murine IL-2R(3 cDNA to be rearranged by of long clones by in situ hybridization under low-stringency condi- sequence of an intracisternal A particle, giving rise to consti- tions (17), using the Xho I fragment of the human IL-2Rp6 tutive expression of the IL-2RB mRNA. cDNA from pIL-2Rf330 (14) as probe. Subsequently, the cDNA from one of the positive clones (i.e., AMIL2R,3-26) Interleukin 2 (IL-2) has been widely recognized as a cytokine was used as a probe to isolate EL-4-derived cDNA clones (lymphokine) that plays a crucial role in regulation of the following standard procedures (17). immune response by exerting multiple biological activities on Expression of the Murine IL-2R13 cDNA. The IL-3- target cells. The best characterized activity of IL-2 is the dependent pro-B line, BO, a subline of BA/F3 was kindly promotion of antigen-specific clonal expansion of T lympho- provided by M. Collins (Chester Beaty Laboratories, Lon- cytes (T cells) (1-4). The T-cell proliferation signal is thought don) (18). For the transfection of murine IL-2Rl3 cDNA, a to be transduced by the interactions of IL-2 with a specific subclone ofthe BO cells, designated BAF-B03, was used (19). cell-surface receptor (IL-2R) complex that manifests high It was cultured in RPMI 1640 medium containing 10o (vol/ affinity to IL-2 [dissociation constant (Kd) 10-50 pM] (2-5). vol) fetal calf serum (FCS) and 20% (vol/vol) WEHI-3B The high-affinity IL-2R consists of at least two distinct IL-2 culture supernatant. The BamHI fragment containing the binding molecules; IL-2Ra (p55) and IL-2RB (p70-75) (4, entire coding region of the murine IL-2RB was isolated from 6-9). Experimental evidence suggests that IL-2Rf but not pMIL2R8-10, which was constructed by ligating (i) the small IL-2Ra is primarily responsible for the IL-2-induced intra- EcoRI/Nde I fragment isolated from pMIL2R,8-36 [the cellular signal transduction (10, 11). In fact, both human and EcoRI site is located within the IAP long terminal repeat murine IL-2Ras have a relatively short cytoplasmic region (LTR)], (ii) the small Nde IlEcoRI fragment isolated from consisting of 13 amino acids, and replacement of this region AMIL2RB-21 (the EcoRI site is located within the cloning site as well as the transmembrane region by the corresponding ofAgtlO), and (iii) EcoRI-cleaved pUC19 plasmid. It was then regions of either insulin receptor or epidermal growth factor inserted into the BamHI-cleaved p1013 containing the mouse receptor does not affect the formation of a functional high- Ick gene promoter (20). The resulting expression plasmid, affinity IL-2R (12, 13). In contrast, the recent cloning of the pLCKmB, was introduced together with the neomycin- human IL-2RP cDNA revealed that the human IL-2RB con- resistance gene, pSTneoB, into BAF-B03 cells by electro- tains a large cytoplasmic region consisting of 286 amino poration (21), and the cells were seeded at 1 x 10' cells per acids, which potentially includes a functional domain(s) for well in a 24-well tray. The neo-resistant clones were selected intracellular signal transduction (14). in the medium described above containing 2 mg of G418 per The murine IL-2 system has been studied in great detail, ml. The resultant neo-resistant clones were examined for particularly in the context of T-cell development, prolifera- their response to IL-2. For the [3Hithymidine uptake mea- tion, and differentiation. From this viewpoint, it was impor- surement, the neo-resistant cells were seeded at 1 x 104 cells tant to elucidate the structure and function of the murine per well in 96-well microtiter trays in 200 ,ul of RPMI 1640 IL- medium supplemented with 10% FCS and 20% WEHI-3B IL-2RB component to gain further insights into such conditioned medium or various- concentrations of recombi- 2-mediated events. In the present study, we report the nant human IL-2 (Takeda, Osaka, Japan) and cultured for 24 molecular cloning, characterization, and functional expres- hr. During the last 4 hr, 1 ,uCi of [3H]thymidine (specific sion of the murine IL-2RB cDNA.§ We also report the 20 1 Ci = 37 GBq; New England Nuclear) constitutive expression of the IL-2RP mRNA in mouse activity, Ci/mmol; lymphoma line EL-4 due to a DNA rearrangement in the Abbreviations: IL-2, interleukin 2; IL-2R, IL-2 receptor; IAP, intracisternal A particle; LTR, long terminal repeat; EPO-R, erythropoi- The publication costs of this article were defrayed in part by page charge etin receptor. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M28052). 1806 Downloaded by guest on October 2, 2021 Immunology: Kono et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1807

was present in each well, and the incorporated radioactivities A 1 2 3 4 5 6 78 9 were measured as described (12). General Procedures. DNA and RNA blotting analyses, DNA sequence analysis, S1 nuclease mapping analysis, plasmid DNA constructions, and Scatchard plot analysis were carried 28S - out following general procedures as described (22-24). 1 8 S - p RESULTS Isolation and Characterization ofthe Murine IL-2Rfi cDNAs. B 1 2 3 4 56 7 8 Phage clones containing cDNA that cross-hybridizes with the human IL-2Rj3 cDNA were isolated by the in situ hybridization procedure from a AgtlO cDNA library prepared by using 28S - the poly(A)+ RNA from Con A-stimulated mouse spleen cells. 18s - Analysis of each cDNA from three independent clones (AMIL2RJ3-12, -21, -26) revealed that the nucleotide sequences from all three cloned cDNAs were completely identical within the overlapping regions (Fig. 1). The corresponding mRNA FIG. 2. Expression of IL-2R/3 mRNA. (A) Expression of the was found to be constitutively expressed in cell lines such as IL-2R13 mRNA in mouse cell lines. Lanes: 1, primary cultured kidney Ml (myelomonocytic leukemia line), CTLL-2 (IL-2- cells; 2, L929 (fibroblast line); 3, MI (myelomonocytic leukemia line); dependent T-cell line), and EL-4 (lymphoma line with pheno- 4, P3U1 (myeloma line); 5, WEHI 231 (B-lymphoma line); 6, 70Z/3 type CD4', CD8-) (Fig. 2A). The mRNA was expressed at low (pre-B-lymphoma line); 7, M12 (B-lymphoma line); 8, CTLL-2 (IL- 2-dependent T-cell line); 9, EL4 (lymphoma line). Total RNA (20 ,g) levels in unstimulated spleen cells and thymocytes and, upon was used in each lane for the analysis. (B) Expression of IL-2R18 stimulation of the spleen cells by Con A, the mRNA level mRNAs in different mouse tissues. Lanes: 1, Con A-stimulated spleen increased -25-fold (Fig. 2B). The expression of IL-2Rf3 cells; 2, unstimulated spleen cells; 3, kidney; 4, thymocytes; 5, heart; mRNA in EL-4 was interesting in view ofour previous finding 6, liver; 7, lung; 8, brain. Total RNA (20 ,ug) was extracted from each that the introduction and expression of the human IL-2Ra tissue or organ obtained from C57BL/6 mouse. The Pvu II fragment cDNA in this cell line resulted in the expression ofhigh-affinity (540 base pairs) of AMIL2Rf-26 was used as a probe (Fig. 1). The IL-2R (24). amount of RNA loaded was calibrated relative to the ribosomal RNA Blotting analysis of the poly(A)+ RNAs from Con A- blotted in each preparation (data not shown). stimulated spleen cells and EL-4 cells revealed that the IL-2Rf mRNA from EL-4 cells was slightly larger than that cDNA contains a large open reading frame that encodes a from spleen cells (data not shown; see below for details). To protein consisting of 539 amino acids. The N-terminal 26 gain further insight into the expression of the IL-2R,8 mRNA residues should correspond to the signal sequence for the in EL-4 cells, we next isolated the corresponding cDNAs. receptor (25) and the subsequent 214, 28, and 271 residues Nucleotide sequence analysis of the two cDNAs (pMIL2R,8- constitute the extracellular, transmembrane, and cytoplas- 22, -36) revealed that they were identical in the sequence to mic regions, respectively (see Fig. 3). The mature IL-2RB those derived from the normal spleen cells except at the 5' polypeptide thus consists of 513 amino acids with the calcu- noncoding region (Fig. 1; see below). lated molecular weight of 57,767. Primary Structure of the Murine IL-2Rfi and Its Homology The overall similarity of the murine IL-2Rf3 to the human to Other Receptor Components. As depicted in Fig. 1, the IL-2Rf3 (14) is 58% (Fig. 3) at the amino acid level, wherein 0 500 1000 1500 2000

2 a o * , .; 31. - .-, 11a D & 0. & -C0 0. z Y Z CLU 0 4. o. 0 X <9 A I I I --LL-N" I I ""1I.I .I I.I I 1 I I II. i:::: -V /

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AMIL2RO3-12 - AMIL2RP-2 1 AMIL2R3-26 1 pMIL2RP-22 pMIL2RP-36

I.------, IAP 5'LTR FIG. 1. Structure ofthe murine IL-2Rf cDNA. Schematic representation ofthe cloned cDNAs. Stippled, hatched, open, and solid rectangles represent the signal sequence, and the extracellular, transmembrane, and cytoplasmic regions, respectively. Broken line in pMIL2Rp-22 and -36 represents the sequence corresponding to the LTR of mouse lAPs. Downloaded by guest on October 2, 2021 1808 Immunology: Kono et al. Proc. Natl. Acad. Sci. USA 87 (1990)

50 100 mL-2RB M--ATIALPWSLSLYVFLLLLATPWASAAV .------KNCSHLE& YNSRANV S WSHEEALNVTTCHVHAKSNLRHWNK L-----.TL hL-2RB M--AAPALSWRLPLLILLLPLATSWASAAV .------NGTSQFT t FYNSRANI WCVWSQDGALQDTSCQVHAWPDRRRWNQ C L-----LP EPO-R MDKLRVPLWPRVGPLCLLLAGA-AWAPSPSLPDPKFESKAALLASRGSEEIL JFTQRLEDL C FWEEAASSGMDFNYSFSYQLEGESRKS LHQAPTVR * 0* 00 ** * 0**000 ** O 00 * * 0 0 0* * 150 200 VRQASWA eLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHS VSQASW' : NLILGA-PDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHT GSVRFW i L--PT-ADTSSFVPLEL-QVTEASGSPRYHRI IHINEV-----VLLDAPAGLLARRAEEGSHVVLRWLPPPGAPMTTHIRYE-VDVSAGNR * * * 00 OQO 0000 0 * 0 0 0 * * 0* 0 0 0 00 0 0 0* * 2 3?0 WEDASVLSLKQRQQWLF EMLIPSTSYEVQVRVKAQRNNTGT-WSPSQP TFRTRPADPMKEILPMS LRYLLLVLGCFSGFFSCVYILVKCRYLGPW WEEAPLLTLKQKQEWIC ETLTPDTQYEFQVRVKPLQGEFTT-WSPWSQ AFRTKPAALGKDTI ILVYLLINCRNTGPW AGGTQRVEVLEG-RTEC SNLRGGTRYTFAVRARMAEPSFSGFWSAWSE ------ASLLTAS--DLDPLILTLSLLLISILLLVLALLSHRRT 0 0 0 0 * * ** 00 *** 0 *00 *0 0 00 350 400 LKTVLKCHIPDPSEFFSQLSSQHGGDLQKWLSSPVPLSFFSPSGPAPEISP--LEV GDSKAVQLLLLQKDSAPLPS-PSGHSQASCFTNQGYFFFHLP LKKVLKCN PDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISP--LEV RDKVTQLLLQQDKVPEPASL-SSNHSLTSCFTNQGYFFFHLP LQQKIWPG PSPESEFEGLFTTHKGNFQLWLLQRDGCLWWSPGSSFPEDPPAHLEV EPRWAVTQAGDPGADDEGPLLEPVGSEHAQDTYLVLDKWLLP * 0 ** * *O**0*** 0 0**00 ** O **** 0 0 0 * 0 * 02* 4R0 5?0 NALEIESCQV-YFTYDPCVEEEVEEDGSRLP------EGSPHPPLLPLAGEQDDYCAFPP-RDDLLLFSPSL----STPNT--AY DALEIEACQV-YFTYDPYSEEDPDEGVAGAP------TGSSPQPLQPLSGEDDAYCTFPS-RDDLLLFSPSLLGGPSPPST--AP RTPCSENLSGPGGSVDPVTMDEASETSSCPSDLASKPRPEGTSPSSFEYTILDPSS-QLLCPRALPPELPPTPPHLKYLYLVVSD------SGISTDYSS 0 * 0 ** 00 * 0 0 0*0 * * 0 0 0 * 0 *0 *0 * 0 GGSRAPEERSPLSLHE------GLPSLASRDLMGLQRPLERMPEGDGEGLSANSSGEQASVPEGNLHGQDQDRGQGPILTLNTDAYLSLQELQAQ GGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQ GGSQGVHGDSSDGPYSHPYENSLVPDSEPLHPGYVACS ------*** 0 0 0 0

DSVHLI (539a.a.) DPTHLV (551a.a.) __ __ _ (507a.a.) FIG. 3. Homology of murine (in) IL-2Rf3 with human (h) IL-2Rj3 and murine EPO-R. The alignment by D'Andrea et al. (26) was slightly modified by including the murine IL-2RB sequence. Asterisks and open circles represent identical amino acids (single-letter code) and conservative substitutions (27), respectively. Gaps (-) were introduced to maximize homology. Thin underlining indicates the boundaries ofthe signal sequences. Transmembrane regions are indicated by thick underlining. The four cysteine residues conserved among the three receptors are boxed. Two strongly conserved segments (I and II) are also boxed. a.a., Amino acids. 7 of9 cysteine residues in the extracellular region, which may clones but none of p1013-transfected clones responded to contribute in folding the ligand binding domain, seem to be IL-2 (1 nM). Subsequent S1 nuclease mapping analysis conserved between the two species. Within the cytoplasmic showed that only the IL-2-responsive clones expressed the region, the sequence homology between the two receptors is mRNA for the transfected gene (results not shown). The rather confined to the upstream (i.e., N-terminal) region, properties ofone positive clone, termed BOM,83, were further suggesting the functional importance of this region in signal examined. As demonstrated in Fig. 4B, expression of the transduction. high-affinity IL-2R (Kd 60 pM; :1000 sites per cell) was Recently, the presence of extensive sequence similarity detected in BOM,3 by Scatchard plot analysis. The high- between the human IL-2Rf3 and the murine erythropoietin affinity IL-2R was not detectable in the parental BAF-B03 receptor (EPO-R) has been reported (26). The amino acid cells (19) as well as in another neo-resistant, pLCKmp3- sequence of the murine IL-2Rp was aligned with those of the transfected clone (BOM,82), which showed no growth re- human IL-2Rf3 as well as the murine EPO-R (28) (Fig. 3). The sponse to IL-2 (results not shown). As expected, IL-2 en- murine IL-2RB shows notable sequence similarities with the hanced the [3H]thymidine uptake by BOM,3 cells, but not by murine EPO-R with -19% identity, suggesting that the BAF-B03 and BOMP2 cells (Fig. 4C). There was no indica- IL-2R,1 and the EPO-R diverged following gene duplication tion that the endogenous IL-2R,8 gene expression was in- (gaps were each counted as one substitution regardless of the duced in the BOMP3 cells, as judged by mRNA S1 nuclease length). This alignment revealed four conserved cysteine mapping analysis (results not shown). Thus, the formation of residues as well as two strongly conserved segments I high-affinity IL-2R and response to IL-2 in the BOMf33 cells (aligned amino acids 219-251) and II (amino acids 310-357) is due to the expression of the transfected cDNA. (Fig. 3). Rearrangement of the IL-2RB Gene in a Lymphoma Line Functional Expression of the Murine IL-2Rj3 cDNA. To EL-4. In EL-4 cells, the mRNA for IL-2Re was expressed demonstrate that the cloned cDNA encodes a functional constitutively (Fig. 2A) and it was slightly larger than the IL-2R/3, the cDNA was linked to the mouse Ick gene pro- spleen cell-derived mRNA as revealed by more extensive moter (depicted in Fig. 4A) in the plasmid vector p1013 (20), mRNA blotting analysis (result not shown). Furthermore, the and the resulting expression plasmid pLCKm/3 was cotrans- nucleotide sequences of two independent EL-4-derived fected together with the neo-resistance gene into BAF-B03 cDNAs were found to be identical to each other but com- cells, a subline of mouse IL-3-dependent pro-B-cell line pletely different from spleen cell-derived cDNAs at the 5' BA/F3 (19). The BAF-B03 cells express endogenous IL-2Ra noncoding region. Inspection ofthis diverged region revealed at a high level (-106 IL-2 binding sites per cell); however, the that the sequence from the EL-4 cDNAs corresponds to the IL-2Rl expression is barely detectable (19). Of 107 cells LTR sequence of IAPs (29). In fact, the homology is 96% transfected either with pLCKmj3 or with p1013, 31 and 29 between the two sequences (without counting the two inser- neo-resistant clones, respectively, were obtained. To exam- tion sequences found in the IL-2RB cDNA), suggesting that ine the expression of the murine IL-2RB cDNA, we first the promoter insertion accounts for the IL-2Rp mRNA tested the transformant clones as to whether they respond to expression in EL-4 (data not shown). DNA blotting analysis recombinant human IL-2, assuming that the cDNA-directed of the IL-2R13 gene from EL-4 cells revealed several unique IL-2RS forms a high-affinity IL-2R in conjunction with the bands in addition to the corresponding positive bands ob- endogenous IL-2Ra and would therefore transduce the IL-2 served with DNAs from normal spleen and thymocytes (Fig. signal(s). Three of 31 neo-resistant, pLCKmo-transfected SA). These results are consistent with the idea that the DNA Downloaded by guest on October 2, 2021 Immunology: Kono et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1809 A A pLCKmo Eco RI Pst I Hind IlIl mouse Ick promoter mouse lL-2R3 cDNA hGH m--- m m a poly(A) 1 2 3 4 5 6 7 8 9 (kb)

6.2- - _ 3.4- 2.7- 500bp 1.9- _ 1.5- *00 B 0. 9- .4| .I MD BOM13 B (b 2 p1 30 High affinity IL-2R: M 1 2 3 45 M C -N 1000 sites/cell *F540 Ii 20-Kd=60pM o 20 1 -502~~~458 0 U.

_ -267 - -234 10 20 30 - -213 Bound IL-2 molecules per cell (x 10-3) m -192 * -184 C

E u -124 BOMP3 a f 0 20 --4

.9Ct -104

EL-4 cells * MRNA IAP ATG ~~~~~~IL-2RP goen Eco RI Pst M13 DNA , probe DNA(482nt)

-- BAF-B03 317d *_ protected DNA(165nt) _i BOMP2 54nt 111 nt normal 0 1 10 102 103 mRNA spleen cells Concentration of IL-2 (pM) ATG Ps gene Il IL-2R3 M13 DNA 3* probe DNA(482nt) FIG. 4. Functional expression of murine IL-2Rf cDNA. (A) protected DNA( I I nt) Construction of a murine IL-2R/3 cDNA expression vector, (nt nucleotides) pLCKm,8. Shaded, open, and hatched rectangles represent the mouse Ick gene promoter, murine IL-2R8 cDNA, and a human FIG. 5. DNA rearrangement in lymphoma line EL-4. (A) Southern growth hormone gene segment containing the poly(A)I addition site, blot analysis of the IL-2R,8 gene in normal mouse (C57BL/6) spleen respectively. bp, Base pairs. (B) Scatchard plot analysis of 125[_ cells, thymocytes, and mouse lymphoma line EL-4 (C57BL/6 origin). labeled human IL-2 binding to the BOM,83 cells. Dotted line indicates Lanes: 1, 4, and 7, genomic DNA from EL-4; 2, 5, and 8, genomic the expression of high-affinity IL-2R. The parental BAF-B03 and DNA from spleen cell; 3, 6, and 9, genomic DNA from thymocytes. IL-2-nonresponsive transfectant clone BOMP2 did not display the DNA (5 A&g) was digested by EcoRI (lanes 1-3), Pst I (lanes 4-6), or high-affinity IL-2R (data not shown). (C) Effect of IL-2 on cell HindIII (lanes 7-9) and subjected to DNA blotting analysis. The EcoRI growth of BAF-B03 transformants. IL-2 response was monitored by fragment (800 base pairs) ofAMIL2R-26 was used as a probe (Fig. 1). the [3H]thymidine ([3H]-TdR) uptake in BAF-B03 cells (o), BOM,83 kb, Kilobases. (B) S1 nuclease mapping analysis ofthe IL-2RB mRNA cells (e), and BOMfi2 cells (v). The [3H]thymidine uptake was transcripts. EcoRI/Pst I DNA fragment [165 base pairs (bp)] contain- measured at various concentrations of human recombinant IL-2. ing 54 bp of IAP sequence and 111 bp of murine IL-2R/3 5' sequence was excised from pMIL2Rp-36 (Fig. 1) and cloned into the EcoRI/Pst rearrangement occurred in one of the IL-2R(3 gene alleles. I-cleaved M13mpl8 vector. Synthetic oligonucleotide (5'-CTGCA- Furthermore, SI nuclease mapping analysis of the mRNA GATGCCCAAGGT-3') was labeled at the 5' end and used as a primer (specific activity, 4.3 x After the revealed that the IL-2Rf transcript in EL-4 cells arose from 103 cpm/fmol). primer-extension reaction, the probe DNA was isolated by digesting with Ava II. One the rearranged gene (Fig. 5B). femtomole of labeled DNA fragment [482 nucleotides (nt)] was hybridized with 40 ,ug of total RNA. The hybridized sample (10 ILI) was treated with S1 nuclease (200 units) at 300C for 40 min and analyzed DISCUSSION by 6% PAGE (containing 42% urea). Lanes: 1, RNA from EL-4 cells; In the present study, we report the molecular cloning and 2, RNA from Con A-stimulated mouse spleen cells; 3, RNA from L929 functional expression of the cDNA encoding the murine cells; 4, no RNA; 5, undigested probe DNA. Lane M, DNA size markers (Hae Arrows indicate the IL-2Rp8. The deduced sequence of the III-digested pBR322). protected primary murine IL- DNA fragments. Scheme for mapping the IL-2R,8 mRNA from EL-4 2Rf3 shows marked homology to the human IL-2Rp, partic- cells and normal spleen cells is shown below. The cap sites of the ularly at restricted regions. Of interest are the highly homol- mRNAs have not been experimentally determined. ogous stretches within the cytoplasmic region. We envisage that such stretches may constitute a functional domain(s) to cule(s). It is interesting that one of the homologous stretches couple with the conjectured downstream effector mole- spanning from residues 317 to 357 corresponds to segment II, Downloaded by guest on October 2, 2021 1810 Immunology: Kono et A Proc. Natl. Acad. Sci. USA 87 (1990) which shows remarkable homology to the murine EPO-R advice and Ms. Y. Maeda for typing the manuscript. We also thank (Fig. 3). Recent expression studies of the mutated human Drs. S. Yonehara, J. F. Bazan, and A. D'Andrea for communicating their results to This work is in part by revealed that a region spanning segment II of prior publication. supported IL-2R,8 cDNAs a grant-in-aid-for Special Project Research, Cancer Bioscience from the human IL-2R/3 is indeed essential for the growth signal the Ministry of Education, Science, and Culture of Japan. transduction in BAF-B03 cells (19). An interesting possibility, yet to be examined further, is that IL-2R and EPO-R may 1. Waldmann, T. A. (1986) Science 232, 727-732. utilize a similar mechanism for intracellular signal transduc- 2. Taniguchi, T., Matsui, H., Fujita, T., Hatakeyama, M., Kashima, N., Fuse, A., Hamuro, J., Nishi-Takaoka, C. & Yamada, G. (1986) Immunol. tion. Comparison of the murine IL-2R,8 with other growth Rev. 92, 121-133. factor receptors revealed yet another interesting homologous 3. Greene, W. C. & Leonard, W. J. (1986) Annu. Rev. Immunol. 4, 69-95. stretch in the extracellular region. In fact, a stretch of amino 4. Smith, K. A. (1988) Science 240, 1169-1176. at the end of segment I 5. Robb, R. J., Greene, W. C. & Rusk, C. M. (1984) J. Exp. Med. 160, acids, WS(P/A)WS(Q/E)P, found 1126-1146. (Fig. 3), is particularly conserved between IL-2R,3 and EPO- 6. Sharon, M., Klausner, R. D., Cullen, B. R., Chizzonite, R. & Leonard, R. Curiously, the 5-amino acid motif, WSXWS (which we W. J. (1986) Science 234, 859-863. refer to as the "WS-motif"), is also found in the correspond- 7. Teshigawara, K., Wang, H.-M., Kato, K. & Smith, K. A. (1987) J. Exp. Med. 165, 223-238. ing region of other receptors, such as murine IL-3 (S. 8. Dukovich, M., Wano, Y., Le thi Bich Thuy, Katz, P., Cullen, B. R., Yonehara, The Tokyo Metropolitan Institute of Medical Kehr, J. H. & Greene, W. C. (1987) Nature (London) 327, 518-522. Science, personal communication), murine IL-4 (30), human 9. Tsudo, M., Kozak, R. W., Goldman, C. K. & Waldmann, T. A. (1987) and human colony- Proc. NatI. Acad. Sci. USA 84, 4215-4218. IL-6 (31), granulocyte-macrophage 10. Le thi Bich Thuy, Dukovich, M., Peffer, N. J., Fauci, A. S., Kehrl, J. H. stimulating factor (32) receptors. These receptors function in & Greene, W. C. (1987) J. Immunol. 139, 1550-1556. cells of hematopoietic lineage, and it is tempting to speculate 11. Siegal, J. P., Sharon, M., Smith, P. L. & Leonard, W. J. (1987) Science that the WS-motif constitutes an interaction site for a com- 238, 75-78. may a role in the function 12. Hatakeyama, M., Doi, T., Kono, T., Maruyama, M., Minamoto, S., mon cell-surface molecule that play Mori, H., Kobayashi, M., Uchiyama, T. & Taniguchi, T. (1987) J. Exp. of these receptors. Another notable feature is the conserva- Med. 166, 362-375. tion of four cysteine residues within the extracellular region 13. Kondo, S., Kinoshita, M., Shimizu, A., Saito, Y., Konishi, M., Sabe, H. of the receptors (Fig. 3). In fact, it has recently been pointed & Honjo, T. (1987) Nature (London) 327, 64-67. factor such 14. Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, out that many of the cytokine/growth receptors T., Miyasaka, M. & Taniguchi, T. (1989) Science 244, 551-556. as IL-2, EPO, IL-4, IL-6, growth hormone, and prolactin 15. Herberman, R. B. (1972) J. Natl. Cancer Inst. 48, 265-271. receptors, share a common motif, CX9_10CXWX2632CX1015C 16. Seed, B. & Aruffo, A. (1987) Proc. Natl. Acad. Sci. USA 84, 3365-3369. X, a nonconserved amino acid) 17. Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., (C, cysteine; W, tryptophan; 729-739. to constitute a Furia, A., Miyata, T. & Taniguchi, T. (1989) Cell 58, (30, 33). Thus, these receptors appear growing 18. Collins, M. A., Downward, J., Miyajima, A., Maruyama, K., Arai, K.-I. new family. & Mulligan, R. C. (1988) J. Cell. Physiol. 137, 293-298. The cDNA-directed IL-2R,8 gave rise to high-affinity IL- 19. Hatakeyama, M., Mori, H., Doi, T. & Taniguchi, T. (1989) Cell 59, 2R in conjunction with the endogenous IL-2Ra in the mouse 837-845. mediated the IL-2-induced cell 20. Garvin, A. M., Pawar, S., Marth, J. D. & Perlmutter, R. M. (1988) Mol. pro-B-cell line BAF-B03 and Cell. Biol. 8, 3058-3064. proliferation signal. It is interesting that recombinant human 21. Doi, T., Hatakeyama, M., Itoh, S. & Taniguchi, T. (1989) EMBO J. 8, IL-2 did not show any binding properties to EL-4 cells, which 1953-1958. express the IL-2RB mRNA constitutively, while the EL-4 22. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning:A the cDNA-directed human Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Har- transformant clones expressing bor, NY). IL-2Ra bound IL-2 with high affinity (24). Furthermore, the 23. Fujita, T., Shibuya, H., Ohashi, T., Yamanishi, K. &Taniguchi, T. (1986) human IL-2Rp expressed in EL-4 cells binds human IL-2 Cell 46, 401-407. with intermediate affinity (Kd 4 nM; see ref. 14). Thus, it 24. Hatakeyama, M., Minamoto, S., Uchiyama, T., Hardy, R. R., Yamada, manifests if to G. & Taniguchi, T. (1985) Nature (London) 318, 467-470. is likely that murine IL-2R/3 little, any, affinity 25. Perlman, D. & Halvorson, H. 0. (1983) J. Mol. Biol. 167, 391-409. the human IL-2. However, we cannot rule out the possibility 26. D'Andrea, A. D., Fasman, G. D. & Lodish, H. F. (1989) Cell 58, that murine IL-2R.8 (but not human IL-2RB) fails to reach the 1023-1024. cell surface in the absence of IL-2Ra in EL-4 cells. On the 27. Dayhoff, M. O., Schwartz, R. M. & Orcutt, B. C. (1978) in Atlas of IL-2Ra and the Protein Sequence and Structure, ed. Dayhoff, M. 0. (Natl. Biomed. Res. other hand, murine IL-2RP forming high- Found., Washington), pp. 345-352. affinity IL-2R can both be cross-linked to the 125I-labeled 28. D'Andrea, A. D., Lodish, H. F. & Wong, G. G. (1989) Cell 57, 277-285. human IL-2, and the association of both chains seems to 29. Mietz, J. A., Grossman, Z., Lueders, K. K. & Kuff, E. L. (1987) J. occur in the absence of IL-2 (21, 34, 35). In view of these Virol. 61, 3020-3029. that the IL-2Ra and IL-2R/3 30. Mosley, B., Beckmann, M. P., March, C. J., Idzerda, R. L., Gimpel, observations, it is possible S. D., VandenBos, T., Friend, D., Alpert, A., Anderson, D., Jackson, J., generate, in cooperation, the high-affinity binding domain for Wignall, J. M., Smith, C., Gallis, B., Sims, J. E., Urdal, D., Widmer, IL-2. Clearly, further work is required to elucidate the M. B., Cosman, D. & Park, L. S. (1989) Cell 59, 335-348. molecular nature of the high-affinity IL-2 binding site. 31. Yamasaki, K., Taga, T., Hirata, Y., Yawata, H., Kawanishi, Y., Seed, is B., Taniguchi, T., Hirano, T. & Kishimoto, T. (1988) Science 241, As revealed from the present study, the IL-2R,3 gene 825-828. rearranged in a lymphoma line EL-4 by the insertion of an 32. Gearing, D. P., King, J. A., Gough, N. M. &Nicola, N. A. (1989) EMBO IAP-derived LTR sequence, resulting in constitutive gene J. 8, 3667-3676. expression of this receptor. Insertion of an IAP LTR se- 33. Bazan, J. F. (1989) Biochem. Biophys. Res. Commun. 164, 788-795. results in aberrant 34. Saragovi, H. & Malek, T. R. (1987) J. Immunol. 139, 1918-1926. quence upstream of the c-mos gene 35. Yamaguchi, A., Ide, T., Hatakeyama, M., Doi, T., Kono, T., Uchiyama, activation of this cellular oncogene in mouse myeloma cells T., Kikuchi, K., Taniguchi, T. & Uede, T. (1989) Int. Immunol. 1, (36). A possible involvement of the IL-2RP in cellular trans- 160-168. formation has been implicated by a number of studies (37- 36. Cohen, J. B., Unger, T., Rechavi, G., Canaani, E. & Givol, D. (1983) the is not in Nature (London) 306, 797-799. 39). It has been reported that IL-2RB expressed 37. Tsudo, M., Goldman, C. K., Bongiovanni, K. F., Chan, W. C., Winton, human CD4', CD8- T lymphocytes unless the cells are E. F., Yagita, M., Grimm, E. A. & Waldmann, T. A. (1987) Proc. Natl. stimulated by mitogen (40). In this regard, our findings may Acad. Sci. USA 84, 5394-5398. be indicative that the promoter insertion and aberrant IL-2RP 38. Tagawa, S., Hatakeyama, M., Shibano, M., Taniguchi, T. & Kitani, T. have a role in the (1988) Blood 71, 1161-1164. gene expression in EL-4 might played 39. Allouche, M., Sahraoui, Y., Augery-Bourget, Y., Ohashi, Y., Sugamura, development of the lymphoma. K., Jasmin, C. & Georgoulias, V. (1989) J. Immunol. 143, 2223-2229. 40. Tsudo, M., Kitamura, F. & Miyasaka, M. (1989) Proc. Natd. Acad. Sci. We thank Drs. C. Weissmann and E. Barsoumian for invaluable USA 86, 1982-1986. Downloaded by guest on October 2, 2021