The EMBO Journal vol.9 no.13 pp.4367-4374, 1990

Molecular cloning and expression of the murine interleukin-5 receptor

Satoshi Takaki, Akira Tominaga, Yasumichi Hitoshi, Seiji Mita, Eiichiro Sonoda, Naoto Yamaguchi and Kiyoshi Takatsu Department of Biology, Institute for Medical Immunology, Kumamoto University Medical School, Kumamoto 860, Japan

Communicated by G.Kohler

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45 284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine [L-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-S binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-S receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified. Key words: cDNA cloning/cytokine receptor family/murine IL-5 receptor/soluble receptor

Introduction IL-5, previously defined as T cell-replacing factor (TRF) (Takatsu et al., 1980, 1990), or B cell growth factor II (BCGFII) (Swain et al., 1983) is an inducible glycoprotein primarily produced by activated T cells (Takatsu et al., 1988; Swain et al., 1988; Tominaga et al., 1988). A cDNA encoding murine (Kinashi et al., 1986) and human IL-5 (Azuma et al., 1986; Yokota et al., 1987) has been isolated, and recombinant IL-5 is now available. IL-5 has been shown to possess a broader spectrum of biological activities. In addition to inducing proliferation and differentiation of murine B cells (Kinashi et al., 1986; Sonoda et al., 1989; Oxford University Press

Tominaga et al., 1989, 1990), IL-5 induces expression of IL-2 receptor (Tac) on B and T cells (Loughnan et al., 1987; Harada et al., 1987; Takatsu et al., 1987; Nakanishi et al., 1988), and promotes growth and differentiation of eosinophils (Yokota et al., 1987; Sanderson et al., 1988; Yamaguchi et al., 1988). To elucidate how IL-5 can mediate multiple functions in various target cells, we attempted to explore a molecular and biochemical characteristics of the receptor. We have shown from the series of binding and cross-linking studies that murine IL-5 binds to a specific cell surface receptor with both high (KD 150 pM) and low affinity (KD 30 nM) and that at least two polypeptides may comprise the functional IL-5 receptor (Mita et al., 1988, 1989). Subsequently, we prepared two monoclonal antibodies (MAbs), designated H7 and T21, which completely inhibit the binding of IL-5 to its receptor (Yamaguchi et al., 1990; Hitoshi et al., 1990). The MAbs recognize a 60 kd cellsurface protein expressed on IL-5 responsive cells but not on IL-5 unresponsive cells. Thus, the 60 kd protein recognized by both H7 and T21 MAbs is presumably a component of the IL-5 receptor. Here we report the isolation of a cDNA encoding an IL-5 receptor utilizing an expression cloning strategy from murine early B cell line with the use of H7 and T21. Recombinant IL-5 receptor expressed on COS7 cells shows similar binding properties and biochemical characteristics to those of the native receptor with low affinity on the surface of murine IL-5 responsive cell lines. Intriguingly, the introduction of the IL-5 receptor cDNA into a murine hematopoietic progenitor cell line resulted in the expression of functional murine IL-5 receptor, indicating that the cloned IL-5 receptor is a component of functional IL-5 receptor with high affinity. We also isolated cDNA clones encoding soluble forms of IL-5 receptor. Analysis of the sequence of IL-5 receptor demonstrates that the IL-5 receptor is a member of a growth factor receptor family (Bazan, 1989). -

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Results Isolation of the munne IL-5 receptor cDNA In order to isolate the cDNA clones for IL-5 receptor, we have applied an expression screening strategy in COS7 cells with the use of two anti-murine IL-5 receptor MAbs, H7 and T21. We constructed a cDNA library (-2 x 106 clones) from a murine IL-5 dependent early B cell line, Y16, in the mammalian expression vector, CDM8 (Seed, 1987). Y16 cells (B220+, Ly-1 +, cytoplasmic Ig-, surface Ig-, la- and FcR+) expressed the most abundant number of IL-5 receptor among the cell lines that we examined. Plasmid DNAs of the cDNA library were transfected into COS7 cells, and cDNA clones giving rise to the expression of the antigenic epitopes were screened by panning procedures using H7 and T21 MAbs. After four cycles of the screening, positive clones were further screened by the panning using

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intact H7 MAbs and Petri dishes coated with F(ab')2 fragments of anti-rat IgG to avoid the selection of cDNA encoding Fc receptor and these screenings were repeated twice. Resulting individual plasmid clones were transfected into COS7 cells and analyzed for H7 binding by flow cytofluorometry, and we finally obtained a clone pIL-5R.8.

To avoid possible rearrangements and mutations of recovered plasmids from COS7 cells, we screened the original and other Y16 cDNA libraries using pIL-5R.8 cDNA as a hybridization probe and isolated several independent cDNA clones (pIL-5R. 13, pIL-5R.2, and pIL-5R.39) (Figure lA). The inserts of pIL-5R.8 and pIL-5R. 13 were sequenced

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Fig. 1. Structure of the murine IL-5 receptor cDNA. (A) Schematic representation and restriction map of IL-5 receptor cDNAs. The coding regions are boxed. The hatched boxes represent the signal sequence; solid boxes represent the transmembrane domain. Broken lines indicate regions lacking in pIL-5R.2 and pIL-5R.39 when compared with pIL-5R.8 and pIL-5R.13. pIL-5R.8, pIL-5R.2 and pIL-5R.39 whose insert cDNAs were primed using random primers were isolated from a library constructed in CDM8 vector. pIL-5R. 13 whose insert was primed using oligo(dT) primer was isolated from a library constructed in AGS-3 vector. (B) Nucleotide sequence and deduced amino acid sequence of the murine IL-5 receptor cDNA. The sequence was deduced after complete DNA sequence analysis of the above described cDNA clones. Nucleotides and amino acids are numbered on the left margin. The first underlined 17 amino acid residues represent the signal sequence. The transmembrane domain is marked by a box. The potential N-linked glycosylation sites are shown by double underlines. The cysteines are marked by asterisks. (C) The deduced amino acid sequence in pIL-5R.2, which occurs following the deletion of nucleotides 986-1164, designated by the open triangle (V) in (B). (D) The deduced amino acid sequence in pIL-5R.39, which occurs following the deletion of nucleotides 986-1079, designated by the closed triangle (V) in (B).

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Murine interleukin-5 receptor gene

by standard procedures (Figure IB). In the composite sequence shown in Figure iB, pIL-5R.8 is represented by nucleotides -302 and 1506 and clone pIL-5R.13 by nucleotides - 165 to 3269. Each sequence contains a large open reading frame that encodes a polypeptide of 415 amino acids (Figure iB). A hydropathy plot of the deduced amino acid sequence predicts two hydrophobic regions. The first hybrophobic region spans the NH2-terminal 17 amino acids and may represent a signal sequence (von Heijne, 1986). The second region (22 amino acids) spans from residues 340-361 and appears to constitute a transmembrane domain of the IL-5 receptor molecule. The predicted mature polypeptide of IL-5 receptor consists of 398 amino acids with a calculated molecular size of 45 284 daltons. There are six potential N-linked glycosylation sites of which four are found in the putative 322 amino acid extracellular domain. Such a post-translational modification may account for the difference of molecular sizes between the estimated mature IL-5 receptor (60 kd) (Yamaguchi et al., 1990) and the calculated one (45 kd). The putative transmembrane domain is followed by a 54 amino acid intracellular domain. The nucleotide sequences of pIL-5R.2 and pIL-5R.39 were identical to that of pIL-5R.8 except that they lacked nucleotides 986-1164 and 986- 1079, respectively. These deletions in two cDNA clones will cause altered translational reading frames that extend for an additional four amino acids (pIL-5R.2) or 62 amino acids (pIL-5R.39) after the end point of deletion, respectively (Figure IC and D). These two predicted amino acid sequences lack the transmembrane domain.

disuccinimidyl tartarate (DST). A major broad band of -97 kd was detected when COS7 cells transfected with pCAGGS-5R were cross-linked with 35S-labeled IL-5 (Figure 3A, lane 3). Under reducing conditions, the size of cross-linked complex decreased to 75 kd (lane 5) due to the dissociation of monomeric 35S-labeled IL-5 from the major cross-linked complex, because biologically active IL-5 binds to its receptor as disulfide-linked dimer (Takahashi et al., 1990). These complexes were disappeared by the addition of excess amounts of unlabeled IL-5 (lanes 4 and 6). By contrast, none of cross-linked band was detected by crosslinking of COS7 cells transfected with vector alone (lanes 1 and 2). Then we carried out the immunoprecipitation by using H7 MAb and cell lysates from cell-surface 125ilabeled COS7 transfectants. H7 MAb immunoprecipitated the 60 kd protein from cell lysates of 125I-labeled COS7 cells transfected with pCAGGS-5R, but not from these transfected with control vector (Figure 3B). The sizes and

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Binding and biochemical characteristics of the cloned murine IL-5 receptor We next performed expression studies of pIL-5R.8 in order to determine whether the cDNA product binds IL-5. To attain higher levels of expression, we also utilized another strong expression vector pCAGGS (kindly provided by Dr Miyazaki), into which the cDNA spanning the entire coding sequence was inserted (pCAGGS-5R). Binding characteristics of the cloned IL-5 receptor expressed on COS7 cells were shown in Figure 2A and B. COS7 cells transiently transfected with pIL-5R.8 and with the expression plasmid pCAGGS-5R were shown to bind with 35S-labeled IL-5 and its binding was competed with unlabeled murine IL-5. The saturation binding curve for 35S-labeled IL-5 binding to pIL-5R.8 transfectants showed only a single class (low affinity) of IL-5 binding with an apparent equilibrium dissociation constant (KD) of 2.0 nM and 1.2 x 104 binding sites per cell (Figure 2A). When COS7 cells were transfected with pCAGGS-5R, they expressed -70 times more IL-5 binding sites (-8.8 x 105 sites per cell) than these on COS7 cells transfected with pIL-5R.8. However, we detected only a single class (low affinity) of IL-5 binding sites with an apparent KD of 9.6 nM (Figure 2B). Untransfected COS7 cells or COS7 cells transfected with vector alone showed no significant binding (data not shown). Y16 cells, the source of cDNA library consistently showed two IL-5 binding sites (high affinity, KD = 20 pM, 1200 sites per cell; low affinity, KD = 5.1 nM, 22 000 sites per cell) (Figure 2C). In order to characterize further the molecular structure of recombinant IL-5 receptor, we performed chemical crosslinking experiments with the use of 35S-labeled IL-5 and

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characteristics of expressed molecules are similar to those observed as a major component (60 kd) of the IL-5 receptor on IL-5 dependent cells (Yamaguchi et al., 1990). We then examined the characteristics of translation products of pIL-5R.2 and pIL-5R.39, both lack nucleotides encoding the transmembrane region. pIL-5R.2 and pIL-5R.39 cDNAs were inserted into pCAGGS vector resulting in pCAGGS-5R.2 and pCAGGS-5R.39, respectively, and transfected into COS7 cells. In the conditioned media of COS7 cells transfected with pCAGGS-5R.2, a 50 kd protein was secreted, and the binding of radiolabeled IL-5 to Y16 cells was inhibited by the conditioned media (data not shown). This result suggests that soluble IL-5 receptor is secreted into extracellular media. The conditioned media of COS7 cells transfected with pCAGGS-5R.39 did not inhibit the binding of IL-5 to Y16 cells. A translation product of a 60 kd protein which is predictable from the cDNA sequence was not detected in the media.

of IL-5 receptors is really observed in cell lines that express the IL-5 receptor, we applied the PCR technique for the amplification of cDNA (Figure 4B). Such analysis revealed the existence of variant transcripts corresponding to both the membrane-bound form (Figure 4B, arrow a) and soluble forms (arrow b,c) of IL-5 receptor in cell lines bearing IL-5 receptor. As shown in Figure 4B, the transcripts for the membrane-bound form appeared to be expressed most abundantly among transcripts for the IL-5 receptor. Unexpectedly, an extra band was detected in IL-5 receptor bearing cell lines (arrow d). This band seems to be derived from the IL-5 receptor transcripts, because corresponding bands could be obtained by using different combinations of primers (data not shown). This type of mRNA might be a product resulting from an alternative splicing of the precursor mRNA for IL-5 receptor.

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Expression of the murine IL-5 receptor transcripts Expression of IL-5 receptor mRNA was examined by hybridization with a cDNA insert from pIL-5R.8 as a probe. The RNA blot analysis revealed the presence of two mRNAs (-- 5.0 kb and 5.8 kb) whose expression was restricted to cell lines (Y16, BCLI-B20 and MOPC104E) (Figure 4A) that have been identified as bearing the IL-5 receptor (Mita et al., 1989). In contrast, the expression of IL-5 receptor mRNA was not detected in IL-5 unresponsive cells, e.g. X5563, FDC-P1, Ltk- and MTH cells. Overall, the level of mRNA expressed in particular cells correlated well with the number of low affinity IL-5 receptor per cell. The relative ratio of 5.0 kb to 5.8 kb mRNA varied among IL-5 receptor bearing cell lines. To analyze whether the mRNA expression of variant types

Expression of high affinity IL-5 receptor on FDC-P1 transfectants FDC-P1 cells were cotransfected with pCAGGS-5R and pSV2Neo, and stable transformants were selected as

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Fig. 4. (A) An RNA blot analysis of IL-5R mRNA. Poly(A)+ RNA (2 ,cg) was subjected to each lane. HindIII-PstI fragment of pIL-5R.8 was used as a probe. Lane 1, Y16; lane 2, BCL1-B20; lane 3, MOPC104E; lane 4, X5563; lane 5, FDC-P1; lane 6, Ltk- and lane 7, MTH. The numbers of low and high affinity IL-5 binding sites per cell were as follows: for Y16, 22 000 and 1200; for BCL1-B20, 7500 and 1000; and for MOPC104E, 18 000 and 50. (B) RNA from the following sources or plasmid DNA was subjected to PCR-based amplification as described in Materials and methods. Lane M, size markers; lane 1, Y16; lane 2, BCLI-B20; lane 3, MOPC104E; lane 4, FDC-P1; lane 5, pIL-5R.8; lane 6, pIL-5R.39; lane 7, pIL-5R.2; lane 8, CDM8 vector. The primer used for PCR reaction (see Materials and methods) define fragments of 548 bp (for pIL-5R.8 type, arrow a), 454 bp (for pIL-5R.39 type, arrow b) and 369 bp (for pIL-5R.2 type, arrow c). Arrow d is an extra band that does not correspond to the fragments derived from the cloned cDNAs.

Murine interleukin-5 receptor gene

described in the Materials and methods. Four transfected clones that were reactive to H7 MAb were established. One of these, FDC-5R was analyzed in detail for IL-5 binding and IL-5 responsiveness compared with these cells transfected with pSV2Neo alone. Figure 5A shows binding experiments with 35S-labeled IL-5 on FDC-5R. FDC-5R cells specifically bound IL-5, whereas FDC-PI cells transfected with pSV2Neo did not (data not shown). The

binding parameters calculated from Scatchard plots derived from the binding data show that FDC-5R cells express 500 binding sites per cell for IL-5 with an apparent KD of 30 pM (high affinity) and 8000 binding sites per cell with KD of 6 nM (low affinity). These are close to the values determined for the IL-5 binding on IL-5 responsive cells. Figure 5B shows that FDC-5R cells became IL-5 responsive for DNA synthesis, whereas neither parental FDC-PI nor FDC-Pl transfected pSV2Neo alone responded to IL-5. -

A Discussion

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To explore the mechanisms of signal transduction induced by various growth and differentiation factors, it is necessary to determine the structure of their receptors. Since receptors for hormones and cytokines including IL-5 are expressed in small numbers on the surface of target cells, isolation and characterization of these receptors have been laborious. Recently, receptors for several cytokines have been molecularly cloned by using various techniques (Sims et al., 1988; Yamasaki et al., 1988; D'Andrea et al., 1989a; Hatakeyama et al., 1989; Mosley et al., 1989; Gearing et al., 1989; Itoh et al., 1990; Goodwin et al., 1990; Fukunaga et al., 1990). To isolate cDNA for IL-5 receptor, we used expression cloning strategy with an antibodymediated cell panning procedure developed by Seed and Aruffo (1987), because we had already prepared MAbs against the receptor for IL-5. Where the cell source of a cDNA library bears abundant numbers of Fc receptors, Fc-mediated binding of MAbs caused a decrease in the efficiency of screening in Seed's cloning system. We experienced just such a situation. To overcome this problem, we modified the panning method with the use of Petri dishes coated with F(ab')2 fragments of anti-rat IgG instead of intact anti-rat IgG. Since the binding of H7 MAb to recombinant Fc receptor expressed on COS7 cells was very loose, H7 MAb easily dissociated from Fc receptor on the cell surface by layering transfectants on 2 % Ficoll in PBS followed by centrifugation during the first antibody separation step of the screening procedure. Introducing this

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B c .12 . Q1 °x 10

c

8-

2

6-

E

4-

2

13IllillMln0.001 0.01

0%

-

0.1 IL-5 (pM)

10

1

Fig. 5. (A) Scatchard plot analysis of 35S-labeled IL-5 binding to the FDC-P1 cells transfected with the cloned cDNA. The inset shows the direct binding data (O, total binding; *, non-specific binding). (B) IL-5 dependent proliferation of FDC-P1 transformants expressing the murine IL-5 receptor. [3H]thymidine incorporation by FDC-P1 cells (0), FDC-P1 cells transfected with pSV2Neo (A) and FDC-P1 cells transfected with pCAGGS-5R and pSV2Neo (A) were determiined as previously described methods (Tominaga et al., 1989). mIL5R

mIL3RI

mIL3RI1 mIL4R mIL7R

hIL2R§ hIL6R

F (llaa) F f Y Y Ll Ll Y[ Q (llaa) TTILILIYa P (llaa) GCLl FY (llaa) (12aa) NiIFIE' F Q I

N T TrIIH (16aa) R- T [lL TILIE NOD (Saa) I ICISIWA NWLQIC Ffi D (Saa) HICISIWIE F E W F T C D (Saa) T E C l Q H L L (5aa) S SF V S QFIT Y N (Saa) S riS C F R K (6aa) V C E W G 49 E L C FFr0 (5aa) VlClFlWlE

128 36 251 31 39 33 118

mEPOR Y hGMCSFR 123 N FIS C F P 28 I HK rPRLR P 53 F TKCR hGHR LJM H mGCSFR 104 N aS

(5aa)

N C T W A

(5aa) K

(7aa) (7aa) (9aa) (7aa) (6aa)

(llaa) V1T AWV P D (llaa) V aaL V|M| K (llaa) (6aa) (llaa) (llaa) F1 Y IIR N (7aa)

(Saa) T C W W N (llaa) SILI TIYS K (7aa) (Saa) S C H W T (13aa) QJ FJ T R (llaa) E (13aa) K S F R S R (llaa) (6aa) V C Q

S K T K T P S RI EI.1 EI7 D

w Q E[j S C E L S E

(7aa) TrA)K WTEITI R (10aa) H RJ Cl V3-PI R C S P V V (10aa) Y RICIS [PV C I P R N (5aa) C v c HIMIE M C L T L N (9aa) TlSl E F L I C E L L P (5aa) W C N LLI L C Q V C Ql L (5aa) F C S L H Q (9aa) F W C S |LZ T C N (6aa) V1G C H K CFC F P D Y K (4aa) N P D JV (4 aa) N S C Y F,N

(5 6aa) Q [i1 E (56aa) E WI S (46aa) HIWIE

(Slaa) TWJN (55aa) KY L

-E]S

(60aa) S W E (56aa) T W Q

V A K K

(53aa) RIWI K (59aa) K W S (57aa) R W E R (61aa) S J K

(2aa) N

SCsai

(53aa)

RI W

L

TM domain mIL5R

(43aa) Ti

S IIQ V RIA A V (6aa) P V

7G1 R

T L (6aa) S G R RTn mIL3RI (53aa) I Y A mIL3RII (46aa) S Y C A R V R! P (4aa) D G I R V R VR S (3aa) T G T mIL4R (51aa) Y Y T A P (5aa) K J F K V R mIL7R (39aa) M Y E V R F P (4aa) F T T hIL2RP (49aa) V R F EP (3aa) Q TE hIL6R (47aa) R T F A V R AR1M (5aa) S G F mEPOR (47aa) R H S V K I (6aa) I L N hGMCSFR (48aa) E PR A rPRLR (48aa) K YL V hGHR (48aa) E YE V mGCSFR (47aa) VY T L

sJ

w Qs L

EW S E W SE W SzW S E W SQ A W S E S W S E CKP D - - H G Y W S RSK Q R N - SIGI N YGE SE C I R (3aa) P GJ F W S P W S P WS W S W S W S W S W S W S

Y (12aa)

WS _-E.EV

X1E Y

(5aa)

P S I (13aa)

P S S (14aa)

P L A (12aa) A (48aa) P A S (9aa) AI E (5aa) S Q12aa) V Y (16aa) G L Q(299aa)

122aa) (26aa) (24aa} 125aa} (25aa} (28aa) ( 22aa} (27aa) (24aa} (24aa}

(24aa}

IS N I K[M L P P VIA (4aa) R r K I P N P S K S L (6aa) T Y R K W K

A RS P L V A I I (6aa) W D Q (6aa) V VVWJflSILPD HLJK T L E Q L (6aa) P WAL' KL K C NT PD PS K E G K T S M H P P Y (6aa) KLLJ RAJ L P E S E F E I W P G IlX (6aa) Q (7aa) R PLIVPPVPIQ IKIDrn LN [5lN P P V P GWK IIKIG F|DIT (7aa) C11 PIVIP. | I JG ILDJP (7aa) L11 (6aa) S§F,'WSDJDAHS S L S S F

Fig. 6. Alignment of the murine IL-5R to cytokine receptors. Murine IL-5R, domains I and II of murine IL-3R (Itoh et al., 1990), murine IL-4R (Mosley et al., 1989), murine IL-7R (Goodwin et al., 1990), human IL-2R beta-chain (Hatakeyama et al., 1989), human IL-6R (Yamasaki et al., 1988), murine erythropoietin receptor (mEPOR) (D'Andrea et al., 1989a), human GM-CSFR (Gearing et al., 1989), rat prolactin receptor (rPRLR) (Boutin et al., 1988), human growth hormone receptor (hGHR) (Leung et al., 1987), and murine G-CSFR (Fukunaga et al., 1990) are aligned. Numbers at the left indicate the amino acid number starting from the first methionine. Identical residues and conserved substitutions are marked by solid boxes and dashed boxes, respectively. Gaps are introduced to maximize homology. 4371

S.Takaki et al.

modification, we succeeded in isolating a cDNA clone of the murine IL-5 receptor. Analysis of the sequence of the IL-5 receptor reveals significant homology in the extracellular domain to several receptors for cytokines, growth hormone and prolactin that have been cloned. The extracellular region of these receptors contains two pairs of cysteine residues and the 'WSXWS' motif located close to the transmembrane domain (Figure 6) (Gearing et al., 1989; Itoh et al., 1990). The murine IL-5 receptor may, therefore, belong to a recently proposed a new receptor gene family (D'Andrea et al., 1989b; Bazan, 1989; Gearing et al., 1989; Itoh et al., 1990) and may have evolved from a common ancestor of other cytokine receptors. The IL-5 receptor has another extra domain from 18Asp to 127Thr preceding the common motif, like GM-CSF receptor (Gearing et al., 1989) and IL-6 receptor (Yamasaki et al., 1988). However, in relation to this extra domain, the IL-5 receptor does not appear to share the structural feature either with GM-CSF receptor or with IL-6 receptor which is composed of Ig-like domain. In contrast to conserved features of extracellular domain, the cytoplasmic domain of the murine IL-5 receptor is considerably unique. The consensus sequences for neither a tyrosine kinase domain nor a catalytic domain of some protein kinases (Hanks et al., 1988) were present in the IL-5 receptor. Moreover, the IL-5 receptor is not composed of the serine-rich region that is observed in fl-chain of IL-2 receptor (Hatakeyama et al., 1989), IL-4 receptor (Mosley et al., 1989), erythropoietin receptor (D'Andrea et al., 1989a), IL-3 receptor (Itoh et al., 1990) and G-CSF receptor (Fukunaga et al., 1990). However, the sequence from 367Leu to 380Asp in the IL-5 receptor following the transmembrane domain contains a proline cluster, which is well conserved among receptors for GM-CSF (Gearing et al., 1989), prolactin (Boutin et al., 1988), and growth hormone (Leung et al., 1987) (Figure 6). The region in the cytoplasmic domain that is homologous among these receptors is likely to have some important role in an association with other membrane components which may be responsible for IL-5 mediated signal transduction or formation of functional high affinity IL-5 receptors. Recombinant IL-5 receptor expressed on COS7 cells displayed the low affinity class of IL-5 binding previously characterized on various IL-5 responsive cells including Y16 cells. The biochemical data unequivocally demonstrate that the cDNA-encoded product is directly involved in the formation of IL-5 receptor. However, this product did not reconstitute the high affinity IL-5 receptor. Intriguingly, FDC-P1 cells transfected with the IL-5 receptor cDNA expressed both high and low affinity IL-5 binding sites and became responsive to IL-5 (Figure 5), although parental FDC-PI cells showed neither IL-5 binding nor IL-5 responsiveness. These results indicate that recombinant IL-5 receptor is really a component of the functional IL-5 receptor with high affinity. We consider that the 60 kd protein encoded by the cloned cDNA may associate with certain cellular protein(s), which may not bind IL-5 by itself, in FDC-P1 cells resulting in the formation of the high affinity IL-5 receptor. We isolated cDNA clones (pIL-SR.2 and pIL-5R.39) that lack the transmembrane portion of IL-5 receptor cDNA. The conditioned media of COS7 cells transfected with pCAGGS-SR.2 but not with pCAGGS-SR.39 inhibited the

4372

binding of IL-5 to IL-5 responsive Y 16 cells, indicating that pIL-5R.2 encodes soluble IL-5 receptor. COS7 cells transfected with both pCAGGS-5R.2 and pCAGGS-5R.39 expressed lesser levels of H7 epitopes on their surface compared with pCAGGS-5R transfectants (data not shown). A small proportion of the products encoded by pCAGGS-5R.2 or pCAGGS-5R.39 may remain on the cell surface of COS7 cells transfected. Although we are unable to detect the translation product of pCAGGS-5R.39 in the cultured media, it is not known whether pIL-5R.39 is functional at this moment. In IL-5 receptor mRNA expression, two classes of mRNAs (-5.0 kb and 5.8 kb) were detected only in IL-5 receptor bearing cell lines (Figure 3). Interestingly, we did not detect 3.5 kb mRNA band in mRNA prepared from Y16 cells. As for the size of cDNA, pIL-5R. 13 cDNA might have primed by possible internal poly(A) sequence residues among 3' non-coding region. As the mRNAs that encode the soluble forms of IL-5 receptor would be only 179 bp (pIL-5R.2 form) or 94 bp (pIL-5R.39 form) shorter than a full-length transcript of IL-5 receptor, it would not account for the difference in size of the two transcripts. We suppose the 5.8 kb mRNA may be the precursor transcript that has to be fully spliced before maturation or may have different polyadenylation sites. It is important to examine whether transcripts for the soluble forms of IL-5 receptor are expressed in the IL-5 receptor bearing cell lines. By PCR technique, we confirmed the transcripts corresponding to the soluble forms of IL-5 receptor lacking putative transmembrane domain in BCLI-B20 and MOPC104E as well as Y16 cells (Figure 4B). These results indicate that the transcripts for the soluble form of IL-5 receptor are not specific to Y16 cells, and not so rare compared with the transcript for membrane-bound forms. The receptors for IL-4, IL-7 and growth hormone have also been shown to be encoded in both membranebound and soluble forms (Mosley et al., 1989; Baumbach et al., 1989; Goodwin et al., 1990). The existence of cDNAs encoding soluble receptors for growth factor may suggest important regulatory roles of soluble receptors in hematopoiesis and immune response. The effect of the soluble forms of IL-5 receptor in IL-5-mediated signal transduction must await further studies.

Concluding remarks The 60 kd protein, molecularly characterized in this study, is able to bind to IL-5 with a single class of affinity (low affinity). The IL-5 receptor has a relatively short cytoplasmic tail and lacks known kinase domains. It is important to note that an additional molecule appears to be involved in the formation of the functional high affinity IL-5 receptor. Availability of the IL-5 receptor cDNA will allow us further investigation of IL-5-mediated signal transduction and identification of an associated molecule(s) for the IL-5 receptor complex.

Materials and methods Reagents and cell lines MAbs H7 (rat IgG2a) and T21 (rat IgGj) against murine IL-S receptor were prepared and purified as described (Yamaguchi et al., 1990; Hitoshi et al., 1990). The IL-5 dependent early B cell line, Y16, was established from BALB/c bone marrow cells as described (Tominaga et al., 1989) and maintained in the presence of 10 pM IL-S in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 50 ItM 2-ME, penicillin

Murine interleukin-5 receptor gene (100 U/mi) and streptomycin (100 Ag/ml). The murine B cell chronic leukemic cell line BCL1-B20 (in vitro line), an IL-2 dependent CTLL (MTH), an IL-3 dependent cell line (FDC-P1) and mouse myeloma cell lines (MOPC104E and X5563) were maintained as previously described procedures (Yamaguchi et al., 1990).

Construction of cDNA libraries Total RNA was prepared from Y16 cells by the guanidium isothiocyanate/CsTFA method (Okayama et al., 1987) and poly(A)+ RNA was selected by oligo(dT)-cellulose column chromatography. Double-stranded DNA was synthesized according to procedures described by Gubler and Hoffman (1983) with modifications using a kit from BRL.Life Technologies. The blunt-end cDNA was ligated with BstXI non-palindromic linkers and cDNA longer than 1.0 kb was isolated by 5-20% potassium acetate gradient centrifugation (Seed and Aruffo, 1987). The size fractionated cDNA was cloned into BstXI-digested CDM8 (Seed, 1987) or pAGS-3 (Miyazaki et al., 1989).

Screening of cDNA libraries A pool of CDM8 library representing -2 x 106 cDNA clones was expressed in COS7 cells and screened by panning method with a mixture of MAbs H7 and T2 1. Two to three days after transfection, cells were incubated with H7 and T21 MAb, layered on 2% Ficoll solution and centrifuged at 200 g for 5 min. After centrifugation cell pellets were resuspended in PBS containing 0.5 mM EDTA and 5% FCS and distributed into panning dishes (60 mm in diameter) that had been coated with purified goat anti-rat IgG (Cappel). Episomal DNA was collected from adherent cells to the dishes and introduced into Escherichia coli. Transformed E. coli were fused with COS7 cells after the treatment with lysozyme, and subjected to a second round of panning. These panning procedures were repeated four times as described (Seed and Aruffo, 1987). Then positive clones were further screened by the panning using intact H7 MAbs and Petri dishes coated with F(ab')2 fragments of anti-rat IgG (Cappel). This screening process was repeated twice. Resulting individual plasmid clones were transfected into COS7 cells by DEAE-dextran method as described (Seed and Aruffo, 1987). Cells were harvested 2 days after transfection and stained with H7 MAb and FITC-conjugated F(ab')2 fragments of goat anti-rat IgG (Cappel). Cells were then washed and analyzed with a flow cytometer (FACScan, Becton Dickinson Immunocytometry Systems). DNA sequencing The cDNA inserts of clones pIL-5R.8, pIL-SR. 13, pIL-5R.2 and pIL-5R.39 were sequenced by the dideoxy chain termination method (Sanger et al., 1977) using the modified T7 polymerase (Sequenase, USB).

Expression of the IL-5 receptor cDNA A mammalian expression vector pCAGGS (provided by Dr Miyazaki, Kumamoto University Medical School) is a derivative of pAGS-3 (Miyazaki et al., 1989), and contains an SV40 origin of replication, a cytomegalovirus enhancer sequence and a chicken ,B-actin promoter followed by a sequence from the rabbit ,B-globin gene (J.-I.Miyazaki, personal communication). The cDNA sequence obtained by digestion of pIL-5R.8 with XhoI was inserted into a unique EcoRI site of pCAGGS using an XhoI linker, resulting in pCAGGS-5R. The coding region of pIL-5R.2 and pIL-5R.39 were inserted to pCAGGS by the same procedures as that of pIL-5R.8. Transfections were performed by DEAE-dextran method. Cells were trypsinized at 8 h posttransfection and re-seeded in Petri dishes. After 2 or 3 day incubation, cells were harvested after a brief treatment with 0.5 mM EDTA and subjected to further analysis. For FDC-PI cells, pCAGGS-5R and pSV2Neo were transfected by electroporation. Stable transformants were selected in G418-containing medium (400 /Ag/ml). RNA blot analysis and colony hybridization Poly(A)+ RNA (2 ttg) prepared from various cell lines were subjected to electrophoresis through 1 % agarose gel containing 2.2 M formaldehyde as described (Sambrook et al., 1989), and transferred to Gene Screen (Du Pont). Hybridization was carried out according to manufacturer's recommendation. Colony hybridization was carried out as described (Sambrook et al., 1989). As a probe, the HindIII-PstI fragment of pIL-5R.8 was labeled with 32p by random primer labeling method using a kit from Pharmacia. RNA detection using PCR For PCR amplification, 1 ug of poly(A)+ RNA was used to generate the first strand cDNA. One fifth of the cDNA reaction mixture was subjected to PCR amplification. Plasmid DNA (10 ng) was used as control. The primers used for PCR reaction were 5'-GCCATTGACCAAGTGAATCC (position 694-7 13) and 5'-GTGGAATTTCCCATGACTTC (position

1222-1241). PCR reaction (in a volume of 50 1l) contains 200 tLM of each dNTP, 1 AM of each specific primer, 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1.5 mM, MgCl2, 0.001% (w/v) gelatin and 1.25 units Taq polymerase (Perkin Elmer Cetus). The conditions were: 1 min at 94°C; 2 min at 56°C; 3 min at 72°C for 25 cycles in a DNA thermal cycler. A portion of PCR reaction was electrophoresed through a 1 % agarose gel.

Binding assay Recombinant murine IL-5 and [35S]methionine labeled IL-5 were prepared according to procedures as described (Tominaga et al., 1988; Takahashi et al., 1990). For the binding assay, COS7 transfectants were harvested, resuspended at 2- 10 x 104 per 100 l1 of the binding medium (RPMI-1640 medium/25 mM HEPES pH 7.2/0.1 % BSA), and incubated with increasing concentrations of 35S-labeled IL-5 at 370C for 10 min (Mita et al., 1988). For Y16 cells and FDC-P1 transformants, cells were harvested and resuspended at 1 x I05 cells and 1 x 106 per 100 141 of the binding medium, respectively, and binding assays were carried out as described above. In all binding assays, specific binding was defined as the difference between total binding and non-specific binding obtained in the presence of 100-fold excess of unlabeled IL-5. The KD and an average number of binding sites per cell were calculated by Scatchard plot analysis of the binding data.

Chemical cross-linking Transfected COS7 cells (- 5 x 106) were harvested, incubated with 35Slabeled IL-5 (4 nM) at 37°C for 10 min in the absence or presence of 100-fold excess of unlabeled IL-5 and subsequently cross-linked with 1 mM DST (PIERCE) at 4°C for 30 min. Cells were then washed and lysed with lysis buffer containing 1 % Triton X-100 in the presence of protease inhibitors (2 mM EGTA, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 10 JIM pepstatin, 10 tiM leupeptin, 2 mM o-phenanthroline, and aprotinin at 200 KIU/ml) as described (Mita et al., 1989). The detergent extraction mixture was subjected to SDS -PAGE with 7.5% polyacrylamide under non-reducing or reducing conditions, and then analyzed by a Bio-Analyzer 100 (Fuji Photo Film Co. Ltd).

Immunoprecipitation and SDS - PAGE Cells (-5

x

106)

were

radioiodinated with the

use

of IODO-BEADS

(PIERCE). After washing with PBS (pH 7.2), cells were lysed using lysis buffer as described in the above section. lodinated cell lysates were then incubated with H7 followed by the incubation with Protein G-coupled Sepharose (Pharmacia) at 4°C for 12 h. The immunoprecipitates were boiled in SDS sample buffer with 5% 2-ME for 3 min and analyzed by SDS-PAGE on 9% polyacrylamide gels. Gels were then analyzed by

a

Bio-Analyzer 100.

Acknowledgements We thank Drs B.Seed and A.Aruffo, and J.-I.Miyazaki for pCDM8 vector and pCAGGS vector, respectively; and Drs J.-I.Miyazaki, T.Kunisada and S.-I.Nishikawa for valauble advice and critical reading of this manuscript. Supported in part by a Grant-in-Aid for Scientific Research and for Special Project Research, Cancer Bioscience, from the Ministry of Education, Science and Culture, and by Special Coordination Funds for Promoting Science and Technology of the Science and Technology Agency, Japan.

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1990; revised on October 4, 1990

Note added in proof The nucleotide sequence data reported here will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number D90205.