Functional murine interleukin 6 receptor with the intracisternal A particle gene product at its cytoplasmic domain. Its possible role in plasmacytomagenesis.

Two species of the cDNAs encoding murine IL-6-R (one is abnormal and the other authentic) have been cloned from a plasmacytoma cell line, P3U1, and BALB/c mouse spleen cDNA libraries. In the cDNA encoding the abnormal IL-6-R, the region corresponding to an intracytoplasmic domain was replaced with a part of the long terminal repeat of the intracisternal A particle gene (IAP-LTR). The authentic IL-6-R consists of 460 amino acids with the domain of the Ig superfamily. The overall homology between murine and human IL-6-R was 69 and 54% at DNA and protein levels, respectively. The extracellular domain after the Ig- like domain of murine IL-6-R was found to have an homology with those of murine erythropoietin R, human IL-2-R beta chain, murine IL-4-R, and human granulocyte-macrophage CSF-R, as in the case of human IL-6-R, and these receptors have been classified as members of the IL receptor family. In P3U1 cells, the expression of the mRNA encoding abnormal IL- 6-R was much higher than that of the mRNA encoding authentic IL-6-R. An IL-6-dependent human T cell line, KT-3, which did not respond to murine IL-6, acquired the responsiveness to murine IL-6 when transfected with the cDNA encoding abnormal IL-6-R, indicating that abnormal IL-6-R lacking a normal cytoplasmic domain can function. Since IL-6 functions as a potent growth factor for murine plasmacytomas, over-expression of abnormal IL-6-R may function as a positive selection element for the development of certain plasmacytomas.

the integration of the IAP gene into the receptor gene may function as a positive selection element for the development of certain plasmacytomas .

Materials and Methods
Cell Line and Cell Culture. An IL-6 dependent-T cell line, , was used for transfection experiments . KT-3 cells were maintained in RPMI 1640 medium supplemented with 10% FCS, penicillin (50 U/ml), streptomycin (50 lug/ml), 2 mM glutamine, 50 tiM 2-ME, and 2 ng/ml of human rIL- 6 . cDNA Library and Screening. cDNA libraries were made utilizing X phage gt10 from poly(A)' RNA isolated from plasmacytoma cell line, P3U1, and spleen cells of a BALB/c mouse, as described previously (22). The P3U1 cDNA library was probed with an Fsp I-Ban I fragment ofthe pBSF2R.236 insert cDNA encoding human IL-6-R (23). Plaques were blotted on Colony/Plaque Screen membranes (New England Nuclear, Boston, MA) and hybridized in I% SDS, 1 M NaCl, 0.05 M Tris HCl (pH 7 .5), 5 x Denhardt's solution, and 200 p.g/ml salmon sperm DNA at 65'C for 16 h and washed in 2 x SSC and 1% SDS at 60'C. To get X P2 and A 301, the libraries were probed with an Eco RI-Bam HI fragment (probe 1) or a Fok I-Hind III fragment (probe 2) ofX Pl cDNA, as indicated in Fig. 1  Transfection Experiments and Proliferation Assay. The Eco RI insert cDNA of X PI was subcloned at an Eco RI site of pM5G expression vector, kindly provided by Dr. W. Ostertag (24). A Kpn I site of X 301 was blunted and replaced with an Eco RI site. The 1,410-bp Eco RI fragment, which contains authentic IL-6-R cDNA lacking a 3' noncoding region, was subdoned at an Eco RI site of pM5G. KT-3 cells were transfected with pM5G containing either X P1 or X 301 insert cDNA by electroporation . Selection of transfectants was started on day 3 after transfection with 750 pg/ml of the antibiotic G418 (Sigma Chemical Co., St. Louis, MO) .
Stable transfectants (2.5 x 103 cells) were cultured in the presence of various concentrations ofmurine rIL-6, kindly provided by Dr. Y. Akiyama (Ajinomoto Co., Kawasaki, Japan) or human rIL-6 (25) for 66 h. The cells were pulsed with 0.5 tLCi [3H]TdR during the last 6 h and harvested on glass filter paper by an automatic cell harvester (Labo Mash Science Co., Tokyo, Japan). The radioactivity was measured with a scintillation counter (Beckman Instruments, Inc., Fullerton, CA) .

Results and Discussion
Murine IL-6-R. cDNA clones were isolated from the cDNA library of P3U1 cells that were derived from a BALB/c plasmacytoma cell line, MOPC21 . Clones that crosshybridized to the human IL-6-R cDNA, pBSF2R.236 (23), were screened under a nonstringent condition by in situ procedure . 15 positive plaques were identified from 7.3 x 105 plaques . One of them, containing the largest insert cDNA, designated X P1 (Fig. 1 a), was further analyzed . The complete nucleotide sequence of the cDNA insert of X PI demonstrated that the cDNA consisted of 1,470 by and contained a large open reading frame that could encode 440 amino acids (Fig. 1 b).
The overall homology with human IL-6-R was 66 and 48% at the DNA and the protein levels, respectively. The hydrophobic regions corresponding to a signal sequence and a transmembrane domain showed much higher homology (74 and 79% at the protein level, respectively). The domain of the Ig superfamily found in the human IL-6-R was also identified in the molecule encoded by the cloned cDNA. All cysteine residues present in the human IL-6-R were found to be conserved in this murine molecule, except for Cys 211 (human). Furthermore, Trp 134 (human) residue and Trp3°s-Ser-X,xxTrp-Ser3°1 (human) sequence were also found at positions 130 and 300-304, respectively. These residues and four Cys residues (at positions 121, 132, 165, and 176 in human IL-6-R) were found to be conserved among murine erythropoietin R, human IL-2-R (3 chain, murine IL-4-R, and human granulocytemacrophage CSFR, and these receptors have been classified as the IL receptor family (26)(27)(28)(29)(30). All data indicated that the X P1 insert cDNA encoded murine IL-6-R . However, the intracytoplasmic domain ofthe molecule encoded by the cloned cDNA had no homology with that of the human IL-6-R . It was found that the nucleotide sequence ofthe cDNA corresponding to the intracytoplasmic domain showed a striking homology (94%) with that of a part of LTR of the IAP gene ( Fig. 1 c).
Northern blot analysis utilizing the cDNA fragment ( Fig . 1 a, probe 1) specific to the extracytoplasmic domain detected two species of mRNA (1.8 and 5 .5 kb) in P3U1 cells (Fig. 2 a). One major mRNA species had a compatible size (1 .8 kb) with the cloned X P1 insert cDNA. But the other mRNA species, the expression ofwhich was much lower than the former one, had a larger size (5.5 kb), compatible with that of human IL-6-R mRNA . Furthermore, the former mRNA species was shown to be hybridized with an IAP-LTRspecific cDNA fragment (Fig. 1 a, probe 2), but the latter mRNA was not (data not shown) . Moreover, only the second species of the mRNA (5.5 kb) was expressed in the spleen, thymus, and liver of BALB/c mouse, as well as other cell lines, such as the T cell lymphoma cell line, EL-4, and the macrophage cell line, P388D1 (Fig. 2 a). The data indicated the presence ofIL-6-R mRNA without the IAP-LTR. To isolate this mRNA clone, cDNA clones were obtained that were hybridized with probe 1 but not probe 2. Under these conditions, one clone (A P2) from the P3U1 cDNA library and 13 clones from the BALB/c spleen cDNA library were obtained. The nucleotide sequences ofA P2 and a representative clone, A 301 obtained from the BALB/c mouse spleen cDNA library, were determined. In both cDNAs, the nucleotide sequence, as well as deduced amino acid sequence corresponding to both an extracellular and a transmembrane region, were found to be identical with those of the X PI cDNA, although the X P2 insert cDNA lacked a part of 5' region ( Fig . 1 a). However, the region corresponding to an intracytoplasmic region, as expected, did not contain the IAP-LTR gene and showed a significant homology with that of human IL-6-R (53% at the protein level) (Fig .  1 b). Therefore, these cDNA clones were concluded to encode the authentic murine IL-6-R and the overall homology between murine and human IL-6-R was 69 and 54% at DNA and protein levels, respectively.
To examine whether the IL-6-R with its intracytoplasmic portion encoded by a part of the IAP-LTR gene can transduce the signals, the insert cDNA of X PI was subcloned at an Eco RI site of pM5G expression vector (24) and transfected to an IL-6-dependent human T cell line, . Human IL-6 induced cell growth of KT-3 cells, but murine IL-6 did not. However, KT-3 cells transfected with either (in) are aligned with that of human IL-6-R cDNA, pBSF2R236 (23) (ie). The deduced amino acid sequence between positions of 1 and 357 of the insert cDNA of X 301 was identical with that of the insert cDNA of X PI (data not shown) . Gaps are introduced for optimal sequence alignment. The signal sequence, the domain similar to the Ig superfamily, and a transmembrane domain are underlined . Identical amino acids are indicated by asterisks. Conserved cysteine residues are boxed. Some of abnormal or authentic murine IL-6-R cDNA clones were found to be defective in three base pairs, resulting in the lack of Ala 4 (data not shown). (c) Comparison of the nucleotide sequences of the 3' region of the insert cDNA of X PI (top lines) and that of the IAP-LTR se- (continued) the X P1 or X 301 cDNA responded to murine IL-6 to proliferate (Fig . 3) . The data indicated that the IL-6-R, in which an intracytoplasmic domain was replaced with the molecule encoded by a part of the IAP-LTR gene, could function and transduce the signals. This was in complete agreement with our previous observation with human IL-6-R that an intracytoplasmic domain was not required for the signal transduction (20) . C 1159 TGTTGGGAGCCGCGCCCACATTCGCCGTTACAAGATGGCGCTGACA-GCTGTGTTCTAAG ++r+ "tt "f!f "fflfr"" !+a!!"""f!!"t"f+rrle+lff "s! "fr "frr» 2993 TGTTGGGAGCCGCCCCCACATTCGCCGTTACAAGATGGCGCTJaACATCCTG TGTTCTAAG 1218 TGGTAAACAAATAATCTGCGCATGTGCCGAGGGTGGTTCTCCACTCCATGTGCTCTGCCT ++m "A"rr "" rl :ef:ffrrr!! " f"f+f ! ":: + t"" !!f! "r+! " 3053 TGGTAAACAAATAATCTGCGCATGTGCCAAGGGTATCTTATGACTACTTGTGCTCTGCCT enhancer 1278 TCCCCGTGACGTCAACTCGGCCGATGGGCTGCAGCCAATCAGGGAGTGACACGTCCTAGG +"" ++mof!!"""ff!!lum+ruu """r:rrrrf r!lfu+ !flfm "» IAP has been demonstrated to act as an insertional element that upregulates (17,19) or downregulates (31,32) the relevant gene expression. IAP-LTR consists of three regions, U3, R, and U5, and is classified into two groups on the basis of the size ofthe R region (33) . IAP-LTR found in the IL-6-R gene showed a striking homology with that found in murine IgE binding factor (34) and contained U3 and R regions in which the enhancer sequence was included (Fig . 1 c). The study of the organization of the human IL-6-R gene showed that a transmembrane domain and an intracytoplasmic domain of human IL-6-R were encoded by different exons (unpublished data). Therefore, the overexpression ofthe IL-6-R mRNA with the IAP-LTR sequence in P3Ul cells could be due to the enhancer activity of the IAP-LTR gene that was supposed to be inserted between the exons encoding a transmembrane and an intracytoplasmic domain ofthe murine IL-6-R gene. Stabilization ofmRNA may be another possible mechanism for the enhanced expression of the rearranged IL-6-R gene with IAP-LTR.
The IAP gene is expressed in early mouse embryos and many murine plasmacytomas (35,36). The possible role of the IAP gene in oncogenesis was suggested in several studies: the insertion of the IAP gene in the c-mos or the IL-3 gene in plasmacytoma and a myeloid leukemic cell line resulted in the activation of the c-mos or the IL-3 gene, respectively (16)(17)(18)(19). Overexpression of IL-6-R may make cells more sensitive to IL-6 (20). Since IL-6 is a potent growth factor for plasmacytomas and deregulated expression of the IL-6 gene has been suggested to be involved in the oncogenesis of plasma cell neoplasias (8), the deregulated expression of the IL-6-R gene could contribute to accelerate the IL-6-induced promotion of the malignant transformation of plasma cells. A unique feature of an IL-6-R system consisting of two molecules, a ligand binding chain and a signal transducer, allowed the IL-6-R containing the molecule encoded by a part of the IAP-LTR gene instead ofthe normal intracytoplasmic domain to function for the transduction of the growth signals in plasmacytomas. Although, the insertion of the IAP-LTR gene was not commonly observed in various plasmacytomas (Fig. 2 b), this rearrangement may provide a positive pressure for the selection in the development of certain plasmacytomas. Concentration of mIL-6 hIL-6

Summary
Two species ofthe cDNAs encoding murine IL-6-R (one is abnormal and the other authentic) have been cloned from a plasmacytoma cell line, P3Ul, and BALB/c mouse spleen cDNA libraries . In the cDNA encoding the abnormal IL-6-R, the region corresponding to an intracytoplasmic domain was replaced with a part ofthe long terminal repeat of the intracisternal A particle gene (IAP-LTR) . The authentic IL-6-R consists of 460 amino acids with the domain of the Ig superfamily. The overall homology between murine and human IL-6-R was 69 and 54% at DNA and protein levels, respectively. The extracellular domain after the Ig-like domain of murine IL-6-R was found to have an homology with those of murine erythropoietin R, human IL-2-R 0 chain, murine IL-4-R, and human granulocyte-macrophage CSF-R, as in the case of human IL-6-R, and these receptors have been classified as members of the IL receptor family.
In P3U1 cells, the expression of the mRNA encoding abnormal IL-6-R was much higher than that of the mRNA encoding authentic IL-6-R . An IL-6-dependent human T cell line, KT 3, which did not respond to murine IL-6, acquired the responsiveness to murine IL-6 when transfected with the cDNA encoding abnormal IL-6-R, indicating that abnormal IL-6-R lacking a normal cytoplasmic domain can function. Since IL-6 functions as a potent growth factor for murine plasmacytomas, overexpression of abnormal IL-6-R may function as a positive selection element for the development of certain plasmacytomas .