Reconstitution of the functional receptors for murine and human interleukin 5.

The murine interleukin 5 receptor (mIL-5R) is composed of two distinct subunits, alpha and beta. The alpha subunit (mIL-5R alpha) specifically binds IL-5 with low affinity. The beta subunit (mIL-5R beta) does not bind IL-5 by itself, but forms the high-affinity receptor with mIL-5R alpha. mIL-5R beta has been revealed to be the mIL-3R-like protein, AIC2B which is shared with receptors for IL-3 and granulocyte/macrophage colony-stimulating factor. We demonstrated here the reconstitution of the functional receptors for murine and human IL- 5 on the mouse IL-2-dependent cell line, CTLL-2. CTLL-2 was transfected with the cDNAs for mIL-5R alpha and/or AIC2B. Only CTLL-2 transfectant expressing both mIL-5R alpha and AIC2B expressed the high-affinity receptor and proliferated in response to murine IL-5. Then CTLL-2 was transfected with the cDNAs for hIL-5R alpha and/or KH97 (beta c), the human homologue of AIC2B. Though beta c did not contribute much to binding affinity of hIL-5R, only CTLL-2 transfectant expressing both hIL-5R alpha and beta c proliferated in response to human IL-5. These results showed that the beta subunit is indispensable in IL-5 signal transduction. We further investigated the function of IL-5-specific alpha subunit in transmitting IL-5 signals. Mutant mIL-5R alpha, which lacks its whole cytoplasmic domain, was transfected into mouse IL-3- dependent cell line, FDC-P1 expressing AIC2B intrinsically. The resulting transfectant did not respond to IL-5, though the transfectant expressed the high-affinity IL-5R, indicating that the cytoplasmic portion of the alpha subunit also has some important role in IL-5- mediated signal transduction.

I L-5 is a glycoprotein produced by activated T cells and mast . cells. IL-5 was originally identified as a cytokine that stimulates proliferation and differentiation of murine (m) I activated B cells, and proved to regulate the production and function of some other hematopoietic cells, such as CD5 + B cells, eosinophils, and basophils (1). In humans, IL-5 acts mainly on eosinophils. IL-5 induces the production ~f eosinophils from bone marrow progenitors and works for survival and priming of eosinophils in vitro. IL-5 expression was observed in many diseases with eosinophilia, suggesting that IL-5 plays important roles in promoting production and function of eosinophils in vivo (2).
From the series of binding and crosslinking studies, we have shown that mIL-5 binds to a specific cell surface receptor (mIL-5K) with both high (Ka = 10-150 pM) and low affinity (Ka = 2-10 nM) and that two distinct membrane 1 Abbreviations used in this paper: h, human; m, murine; R, receptor. proteins, a and 3, comprise the functional mlL-5K (3). The cloning of mlL-5Rot cDNA discloses molecular constitutions of mlL-SR (4). The mlL-5Ko~ alone binds IL-5 with low affinity. The mlL-5RB does not bind IL-5 by itself, however, it forms the high-affinity IL-5R in combination with the mlL-5Kot (4,5). The mlL-SP,,8 was then identified as AIC2B (6), the homologous protein of the low-affinity mlL-3K (AIC2A [7]) (8,9). The high-affinity mlL-5K is reconstituted on L cell transfectant coexpressing mlL-5Kot and AIC2B. Because the L cell transfectant does not respond to mlL-5 in DNA synthesis, it remains, however, unclear whether the reconstituted high-affinity mlL-5R transduces the IL-5-mediated signal into cell interior.
In contrast to mlL-5K, there are controversial reports about human (h)IL-5R. In many cases, only a single class of hlL-5R with high affinity is detected on hlL-5-responsive cells, but its binding affinity to hlL-5 has been reported as various values (10)(11)(12)(13). There is one report describing two kinds of hlL-5Rs on human erythroleukemic cell line, TF-1 (14). Some reports show that hlL-SR is specific for hlL-5 (10-13), however, others show that binding of hlL-5 is competed by hlL-3 or hGM-CSF at 4~ (cross-competition) (15). We isolated hlL-SRo~ by cross-species hybridization (16). The hlL-SRa resembles mlL-5Ra in its primary structure. It is, therefore, very likely that functional hlL-5R requires the B subunits, and that is the human homologue of AIC2 proteins, KH97, the common B subunit (/3c) of hGM-CSF (receptor) R and hlL-3R (17,18). Recent cloning of cDNAs for mlL-3Ra and mGM-CSFRa show that AIC2B is the common/3 subunit of the receptors for raiL-3 and mGM-CSF (and that AIC2A is mlL-3R~) (19,20). However, COS7 cells transfected with hlL-5Ro~ cDNA binds hlL-5 with almost the same affinity compared to normal eosinophils (16). It remains an unsolved issue how putative hlL-5R~3 contributes to the binding of hlL-5 and transmitting signals.
In this report, we demonstrate the reconstitution of the functional receptors for m and hlL-5 on a mouse IL-2-dependent cell line, CTLL-2. In both mouse and humans, the functional IL-5R complex contains two different membrane proteins, ol and/3, hlL-SR/3 is revealed to be the common molecule that is shared with hlL-3R and hGM-CSFR, and does not contribute much to increasing the binding affinity of hlL-5 in contrast to the mlL-5R system. In addition, using mutant mlL-Sa which lacks the whole cytoplasmic domain, we showed that the o~ subunit has an important role not only in determining the binding specificity, but also in transmitting the growth signal of IL-5.

Materials and Methods
Cells and Reagents. A mouse IL-2-dependent CTLb2 cell line was maintained in RPMI 1640 medium supplemented with 10% FCS and 50/zM 2-ME and 5% conditioned medium from Con A-stimulated rat spleen cells. A mouse ID3-dependent FDC-P1 cell line was maintained in RPMI 1640 medium supplemented with 5% FCS, 50 #M 2-ME, and 5 U/ml of mlL-3, mlb5 was prepared and purified using anti-mlb5 mAb-coupled beads (3). Purified hlb5 was obtained from DNAX Research Institute.
Plasmid Construction. The cDNA fragments were inserted downstream of the SRa promoter in the expression vector, pME18, the derivative of pCEV4 (7) (Maruyama, K., and A. Miyajima, unpublished results). Either neomycin or hygromycin-resistant selection maker was added to the vectors. To construct the plasmid that expresses the truncated mutant mlb5R.c~, an XhoI-PstI fragment of mllc5Ra cDNA corresponding mostly to the extracellular and transmembrane portion was excised, and then ligated into the XhoI and NotI armed pME18 vector, to which the bygromycin-resistant gene had been added, by the use of asymmetric adopters (5'GAG-TGTGTTAGC and 5'GGCCGCTAACACACTCTGCA) that generate a new stop codon. Resulting phsmid-encoded mutant mlL-51~c~ terminated to the end of transmembrane domain (Cys, position 361 in mlL-SRot [4]).
11_,5 Binding Assay. Purified hIL-5 was iodo labeled using IODO-GEN | (Pierce Chemical Co., Rockford, IL). mII~5 were biosynthetically 3SS-methionine labeled, as previously described (3). Specific activities of nSI-labeled hIL-5 and 3SS-labeled mIL-5 were 5.5 x 106 cpm/pmol and 4.7 • 106 cpm/pmol, respectively. For binding assay, 5-10 x 10 s cells were incubated in the binding medium (RPMI 1640 medium containing 25 mM Hepes, pH 7.2 and 0.1% BSA) with increased concentrations of radiolabeled IL-5 at 4~ for 2 h. Cell-associated radioactivity was separated from flee ligand by centrifugation through 3:2 dibutyl/dioctyl phthalates and counted. Specific binding was defined as the difference between total and nonspecific binding obtained in the presence of 100-fold molar excess of unlabeled IL-5. Binding data were analyzed by Scatchard analysis using the EBDA and LIGAND computer programs (Elsevier-BIOSOFT, Cambridge, UK).
Chemical Crosslinking. Crosslinking experiments using mIL-5R transfectants were performed according to the method described previously (3). In brief, 5 x 104 cells were incubated with radiolabeled IL-5, collected, and resuspended in 500 #1 of HBSS containing 1 mM disuccinimidyl tartarate (DST; Pierce Chemical Co.). After incubation at 4~ for 30 rain, cells were solubilized with lysis buffer (PBS containing 1% Triton X-100 and protease inhibitors). After removing insoluble fraction by centrifugation, cell lysates were subjected to SDS-PAGE (7.5% polyacrylamide) under reducing condition. Gel was fixed and dried, then analyzed by Fijix BAS2000 Bioimaging Analyzer (Fuji Photo Film, Tokyo, Japan).

Reconstitution of the Functional hlL-5R. To clarify whether
tic is necessary for the signal transduction through IL-5R, the responsiveness of the transfectants was examined. As shown in Fig. 4, neither CTLL-hSRc~ nor CTLL-flr respond to hlL-5. Only CTLL-hSRa-fl~ responded to hlL-5 in DNA synthesis. Moreover, CTLL-h5Rot-flc acquired the ability to proliferate in response to hlL-5 without IL-2 for more than 2 mo (data not shown). From these results, it was confirmed that tic is indispensable for the functional hlL-5R. In murine IL-2-dependent cells, reconstituted hlL-SR could associate to murine cytoplasmic proteins that transduce growth signals.

Consequences of the Deletion of the Cytoplasmic Domain of
at Subunit. From these transfection experiments, it was confirmed that the common/3 subunit shared among receptors for IL-5, IL-3, and GM-CSF, AIC2B in mice and 3c (KH97) in humans, is indispensable to construct functional IL-SRs.   Sharing of the signal-transducing molecule explains many overlapping biological activities of these cytokines, particularly on eosinophils. However, IL-5, IL-3, and GM-CSF induce different responses depending on cell types. We hypothesized that the signals specific for respective cytokines may exist and be mediated by the relevant ol subunit, specific components to respective receptors. To examine the mlL-SRc~ function, we constructed the mutant cDNA which lacks the whole cytoplasmic domain (Fig. 5 A), and transfected into mouse IL-3dependent FDC-P1 cells endogenously expressing AIC2B. First, we examined interaction of mutant ol subunit and AIC2B. As shown in Fig. 5 B, the high (Kd = 10 pM) and low-affnity (Kd = 2 nM) mlL-5Rs were reconstituted on FDC-P1 transfectants. The high-affnity receptor was detected on FDC-P1 transfectants not only at 4~ but also at 37~ (data not shown), indicating that mutant mlL-5Ro~ molecules were retained on ceU surface and interacted with AIC2B. Though the number of high-affnity mIL-5R on mutant mIL-5Rc~ transfectants was smaller than that on intact mIL-5Ro~ transfectants, it was indicated that the cytoplasmic portion of mIL-SRol is not indispensable to interact with AIC2B. This interaction was also confirmed by crosslinking experiment (Fig. 5 C). The ~75-kD complex of mutant mlL-5RoL and radio-labeled IL-5 and the '~150-kD complex of AIC2B and IL-5 were detected on the FDC-P1 transfectants expressing the mutant c~ subunit. We next investigated the IL-5 responsiveness of mutant mIL-5Rol transfectants (Fig.  6, D). FDC-P1 transfected with wild-type mIL-5Rc~ prolifer- ated in response to raiL-5 as previously shown (4). In contrast, FDC-P1 transfectants expressing mutant mlL-SRc~ did not respond to mlL-5. We obtained essentially the same result with CTLL-2 transfectants. CTLL-2 transfectants coexpressing mutant mlL-SRoL and AIC2B did not respond to raiL-5 (Fig. 6, O). The IL-5-mediated growth signal was never transmitted through the high-affinity IL-SR that consisted of mutant c~ and wild-type/3 subunit, indicating that the cytoplasmic domain of IL-5Rcr has some important roles in transmitting IL-5 signals.

Discussion
We showed that both the ot and/3 subunits are indispensable to form the functional IL-5K. hlL-SRB was revealed to be the same molecule to the common B subunit (jSc) of receptors for hGM-CSF and hlL-3. In contrast to mlL-SR, or to receptors for hlL-3 and hGM-CSF, Bc did not contribute much to hlL-5 binding affinity. We observed the same result using L cell or COS7 cell transfectants (data not shown), indicating that the same affinity of the reconstituted hlL-5R is independent of the nature of the parental cell line used for transfection. In other words, the hlL-5 binding is fully reconstituted by coexpressing hlL-SRot and ~c. Tavenier et al. (24) have also reported that/3c is hlL-5RB. They isolated cDNAs encoding the soluble form of hlL-5Rol, and reported that COS cells expressing chimeric human-mouse IL-5Rot bind hlL-5 with low affinity (Ka = 1 nM) by itself, and that cotransfection of Bc with the chimeric IL-SRc~ leads to a fourfold increase (Kd = 250 pM) in binding affinity (24). The usage of the chimeric IL-SRo~ may lead to somewhat different results from ours presented in this report. Recently, Tavernier et al. (25) reported that hlL-5 binding affinity of COS cells coexpressing the fuU-length hlL-5Rc~ and ~8r is only twofold higher than that of COS cells expressing hlL-5Rc~ alone. However, it is still not clear how the crosscompetition of hlL-5 binding by hlL-3 or hGM-CSF occurs. It is supposed that binding of hlL-5 is irrelevant to the existence of Be, as the binding affinity of ot alone does not differ so much from that of c~/B complex in contrast to hlL-3R and hGM-CSFR systems. The establishment of transfectant coexpressing ~c and ot subunits of hlL-5R, hlL-3R, and hGM-CSFR will answer this confusing question.
In addition to receptors for IL-5, IL-3, and GM-CSF, sharing of signal-transducing component has been demonstrated in receptors for IL-6, leukemia inhibitory factor (LIF), ciliary neurotropic factor (CNTF), and oncostatin M (26). In the IL-6R system, the cytoplasmic and transmembrane domains of the ligand binding subunit (p80 IL-6R) are not required to transduce the growth signal, since the complex of the soluble form of pS0 IL-6R and IL-6 associates with the signaltransducing subunit, gp130. There exist some differences between the IL-5R and receptor systems that use gp130 as a signal transducer. First, not only extracellular but also transmembrane regions of IL-5Rot are necessary to interact with B subunit. The anchoring of IL-5Rol is necessary for interacting with the/3 subunit, since the soluble form of mlL-5Ra inhibits binding of raiL-5 to IL-5-dependent cells (27) 1527 Takaki et al. and does not induce proliferation of FDC-P1 even in the presence oflL-5 (Kikuchi, Y., S. Takaki, and K. Takatsu, unpublished data). Second, the cytoplasmic domain of IL-5Rot plays an important role in transducing the IL-5 signal. Mutant IL-5Rot, which lacks the whole cytoplasmic domain, did not transduce the IL-5-mediated growth signal, though the interaction to AIC2B was not completely impaired (Figs. 5 and 6). It is not clear at this time, how the c~ subunit functions in signal transduction. Each c~ subunit of IL-5R, IL-3R, and GM-CSFR has conserved region (RLFPPX/vPxxxK/RxxL/ ixD) just underneath the transmembrane domain (4,18). This region may be important to interact with the cytoplasmic domain of the common/3 subunit or with a certain common signal-transducing molecule. The elements of the COOHterminal region of the respective ol subunit diversified relatively from one another. The specific function of respective cytokine may depend on the structure of the distal part of the cytoplasmic portion of the c~ subunits. Or, each c~ subunit may simply support the function of the common/3 subunit by inducing conformational change or dimerization of the subunit. CTLL-2 cells transfected with the chimeric receptor that consists of the extracellular domain of erythropoietin receptor (EPO-R) and the cytoplasmic domain of AIC2A, become responsive to EPO (28). Recent studies showed that EPO-R may be activated by homodimerization (29). If that is the case, homodimerization of the cytoplasmic domain of AIC2A may be sufficient to transmit the IL-3-mediated signal. From a high degree of homology between AIC2A and AIC2B (6), it is supposed that similar dimerization mechanism may be involved in IL-5-mediated signal transduction. However, this may only be the case in the signal transduction for cell growth. In other cases, such as signals for B cells to induce differentiation for Ig-secreting cells, the cytoplasmic domain of the ot subunit may have some important roles in transmitting IL-5-specific signal. It is possible that there are several signal-transducing machineries that connect with the membrane receptors to the intracellular device, and that some machineries function only in a limited cell type.
A recent study (30) showed that the cytoplasmic region of ~/c has critical function in transducing growth signal. In addition, it was shown that the cytoplasmic region of hGM-CSFRot also has some roles in transmitting hGM-CSF-mediated signal (30). The transfectant ofmlL-3-dependent Ba/F3 or mlL-2-dependent CTLL-2 expressing mutant hGM-CSFRoL and 3c does not respond to hGM-CSF at the beginning of culture, but adapts to response to hGM-CSF several days later (30). In the case of mlL-5Rol, the cytoplasmic domain was indispensable for transmitting signal, because the FCD-P1 transfectant expressing mutant IL-5RoL and the CTLL-2 transfectant coexpressing mutant IL-5Rol and AIC2B never became responsive to mlL-5. Even when the transfectants were cultured for several weeks in the presence ofmlL-5, no IL-5-responsive transfectant grew up (data not shown). This discrepancy between hGM-CSFR and mlL-5R may suggest the functional difference of each ~ subunit. In addition, CTLL-2 transfectants expressing hlL-3Rot became responsive to hlL-3 because of the induction of the intrinsic AIC2B expression, whereas the transfectant expressing hGM-CSFRot