IL-5 Receptor

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IL-5 Receptor Christopher J. Bagley1,2,*, Jan Tavernier 3, Joanna M. Woodcock 4,2 and Angel F. Lopez 4,2 1

Protein Laboratory, Hanson Centre for Cancer Research, Frome Road, Adelaide, SA, 5000, Australia 2 Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA, 5000, Australia 3 Department of Medical Protein Research, Flanders' Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium 4 Cytokine Receptor Laboratory, Hanson Centre for Cancer Research, Frome Road, Adelaide, SA, 5000, Australia * corresponding author tel: ‡61-8-82223714, fax: ‡61-8-82324092, e-mail: [email protected] DOI: 10.1006/rwcy.2000.20002.

SUMMARY The receptor for IL-5 is comprised of two chains: an  chain that binds IL-5 with moderate affinity but alone is unable to mediate signaling, and a chain that represents the major signaling component of the receptor. The chain is unable to bind IL-5 alone, but when expressed together with the IL-5 receptor  chain (IL-5R) a high-affinity receptor is formed (Kd ˆ 0.15 nM). The chain is shared with the related receptors for granulocyte±macrophage colony-stimulating factor (GM-CSF) and IL-3 and many of the events in IL-5-induced signal transduction are paralleled in the GM-CSF and IL-3 receptors. The IL-5R is expressed on a restricted range of cell types, principally eosinophils, basophils, and their immediate precursors confining the actions of IL-5 to these cells. Thus, the IL-5 receptor is considered to be a target in the development of treatments for allergic inflammatory diseases such as asthma.

BACKGROUND

Tavernier et al. (1991) who also identified the chain of the GM-CSF receptor ( c, cloned by Hayashida et al., 1990) as a component of the IL-5R.

Alternative names IL-5R  chain: IL-5R, Cdw125. IL-5R chain: IL-5R , Cdw131, common chain of the GM-CSF, IL-3 and IL-5 receptors, c.

Structure The IL-5 receptor consists of two chains, denoted  and . The  subunit has specific ligand-binding activity whereas the subunit does not bind IL-5 by itself but enhances the binding of IL-5 and provides the major determinants of signaling capacity of the receptor. The subunit is also a component of the high-affinity receptors for GM-CSF and IL-3.

Discovery

Main activities and pathophysiological roles

The two components of the IL-5R were first identified in crosslinking studies (Mita et al., 1989). The cDNA for IL-5R was cloned by Takaki et al. (1990) and by

Stimulation of the IL-5 receptor in vivo results in the stimulation of production of eosinophils and basophils which exacerbates allergic inflammation.

1904 Christopher J. Bagley, Jan Tavernier, Joanna M. Woodcock and Angel F. Lopez

GENE

Accession numbers GenBank: Human IL-5R: M75914, M96651, M96652, X61176, X61177, X61178, X62156 Human IL-5R : M59941, M38275

Sequence Both the murine IL-5R gene (Imamura et al., 1994) and IL-5R gene (Gorman et al., 1992) have been mapped.

PROTEIN

Accession numbers See Table 1.

Description of protein The nucleotide sequence of the human IL-5R cDNA predicts a polypeptide of 420 amino acids. It is characterized by a 20 residue N-terminal signal peptide, followed by a 322 amino acid extracellular domain, a membrane anchor spanning 20 residues, and a 58 amino acid cytoplasmic tail. The predicted molecular mass for the  chain is 45.5 kDa, indicating that N-linked glycosylation of one or more of the six potential N-glycosylation sites (and perhaps O-glycosylation) contributes to the apparent molecular mass of 60 kDa. The biological role of glycosylation remains unresolved since deglycosylation appears to lead to loss of IL-5 binding (Johanson et al., 1995) although

IL-5R produced in Escherichia coli has been reported to bind IL-5 with near normal affinity (Monahan et al., 1997). The extracellular part of the protein folds into three fibronectin type III structural domains, each having a seven sheet scaffold. The juxtamembrane domain contains a canonical Trp-Ser-Xaa-Trp-Ser motif (WSXWS box) and forms together with the central domain, which itself is characterized by four conserved cysteines, a so-called cytokine receptor module (CRM) (Goodall et al., 1993) also described as a cytokine-binding domain (CBD). The N-terminal domain is more related to the WSXWS box domain (Tuypens et al., 1992), and contains two Cys residues, which may form an intradomain disulfide bond (Cornelis et al., 1995a). With the exception of a Box-1 motif (Pro-Pro-Xaa-Pro), no consensus sequences are found in the cytoplasmic domain. Ligand recognition involves residues in the hinge between the central and membrane-proximal domains as well as a region in the N-terminal domain (Cornelis et al., 1995b). The nucleotide sequence of the human IL-5R cDNA predicts a polypeptide of 897 amino acids. The protein is characterized by a 16 residue N-terminal signal peptide, followed by a 422 amino acid extracellular domain, a membrane anchor spanning 22 residues, and a 437 amino acid cytoplasmic tail. The predicted molecular mass for the chain is 97.3 kDa, indicating that N-linked glycosylation and probably O-glycosylation contribute to the apparent molecular mass of approximately 130 kDa. The extracellular part of the protein folds into four fibronectin type III structural domains grouped into two CRMs (Bagley et al., 1997). The membrane-proximal CRM contains the main determinants for interaction with IL-5, principally in the loops between the B and C and F and G strands of domain four (Woodcock et al., 1994, 1996). The cytoplasmic domain contains a Box-1 motif (Pro-Pro-Xaa-Pro) responsible for

Table 1 Protein accession numbers ID (Swiss)

AC (Swiss)

Human IL-5R

IL5R_HUMAN

Q01344

Mouse IL-5R

IL5R_MOUSE

P21183

Guinea pig IL-5R

U55215

Human c

CYRB_HUMAN

P32927

Mouse c

CYRB_MOUSE

P26955

Guinea pig c Rat IL-3-specific (or common) chain

AC (GenPept)

U94688 Q64146

IL-5 Receptor 1905 interaction with JAK kinases, eight tyrosine residues that are susceptible to phosphorylation, and a recently reported phosphoserine-containing site that binds 14-3-3 proteins (Stomski et al., 1999).

Relevant homologies and species differences Both chains of the IL-5R are members of the cytokine receptor family. The IL-5R is most closely related to the GM-CSFR and IL-3R and more distantly related to the IL-13R2. The two CRMs of c are not closely related to each other, nor to other members of the cytokine receptor family. Apart from the immediate membrane-proximal region, the cytoplasmic domain of c is not significantly related to those of other cytokine receptors. Human and murine IL-5R  chains are 68% identical (extracellular 71%; intracellular 58%). Human and murine common (GM-CSF and IL-5 receptor) chains are 56% identical (extracellular 57%; intracellular 55%).

Affinity for ligand(s) The IL-5R subunit is ligand-specific and binds with intermediate affinity in humans (Kd ˆ  0.5±1 nM) and low affinity in the mouse (Kd ˆ  5±10 nM). Upon association with the subunit, similar high-affinity binding (Kd ˆ 150 pM) is observed for both species. The subunit does not have any detectable affinity for IL-5 by itself (Takaki et al., 1990, 1991; Devos et al., 1991; Tavernier et al., 1991; Murata et al., 1992). The dissociation rate of mIL-5 from the / complex is considerably slower (t1=2 > 1 hour) than from the low-affinity -binding site (t1=2 < 2 min) (Devos et al., 1991). IL-5 binds to its receptor with unidirectional species-specificity: mIL-5 binds with comparable affinities to both murine and human  subunits, but hIL-5 displays 100-fold lower binding affinity for the murine  chain, compared with its human counterpart, a cross-species pattern that is mirrored in their biological activities (Lopez et al., 1986).

Cell types and tissues expressing the receptor Expression of the IL-5R in humans is most prominent on eosinophils and basophils (Lopez et al., 1991). Its appearance on multipotential myeloid progenitors is critical for their development towards the eosinophilic lineage. It remains expressed on mature cells, but the

expression level is controlled at different levels. In the mouse, in addition to these cell types, the IL-5R is also found prominently on B cells belonging to the B1 lineage (Hitoshi et al., 1990). Although there has been no report of direct measurement of IL-5R on human B cells, IL-5 appears to augment terminal differentiation of mitogen-stimulated B cells in some cases.

Regulation of receptor expression Two promoters, P1 and P2, have been identified in the hIL-5R gene (Sun et al., 1995; Zhang et al., 1997). P1 and P2 precede the first and the second exon respectively, and show no significant sequence similarities. P1 is myeloid and eosinophil lineagespecific, whilst the use of P2 is restricted to eosinophilic HL60-C15 cells. It is at present unclear whether differential use and regulation of both promoters occur during eosinophilic differentiation. The P1 promoter contains multiple consensus binding sites for AP-1, C/EBP, GATA, and PU.1. In addition, the region between ÿ432 and ÿ398 was shown to contain a unique cis-element (EOS1), necessary and sufficient for the expression in eosinophilic cell lines (Sun et al., 1995). The putative myeloid- or eosinophil-specific binding factor(s) has not been identified so far. Involvement of the adjacent AP-1 element at position ÿ440 to ÿ432 in the expression in eosinophilic HL-60 cells has been demonstrated (Baltus et al., 1998), suggesting cooperation between the cognate binding transcription factors. Supershift analysis experiments showed the presence of cJun, CREB, and CREM in the AP-1-binding complexes. The P2 promoter sequence shows the presence of AP-1, C/EBP, GATA, CLEO (IL-5), and its consensus binding sites. A unique functional motif was identified between positions ÿ19 and ÿ14. It is involved in the binding of a hitherto unidentified eosinophilic HL-60-C15-specific transcription factor(s). Alternatively, it may also serve as a noncanonical TATA box, since such a motif is lacking in the P2 promoter. Rapid downregulation (maximum inhibition within 2 hours) of the hIL-5R mRNA is induced in peripheral blood eosinophils upon treatment with IL-3, IL-5, or GM-CSF. In contrast, similar treatment leads to upregulation of the IL-3R, GM-CSFR, and c mRNA. The mechanisms involved were shown to be promoter activation and reduced mRNA degradation, respectively, indicating differential regulation (Wang et al., 1998). It is at present unclear whether the downmodulation occurs for P1, P2, or both. The promoter of the mIL-5R contains consensus binding sites for AP-1, GATA, NF-IL-6, NFB, and

1906 Christopher J. Bagley, Jan Tavernier, Joanna M. Woodcock and Angel F. Lopez SP-1. Little is known about transcription factors controlling cell type-specific expression of the mIL5R chain.

Release of soluble receptors Human eosinophils express through alternative splicing different transcripts from the same IL-5R locus (Tavernier et al., 1992; Tuypens et al., 1992). As a result, in addition to the membrane-anchored receptor, two soluble isoforms can be produced. One of these soluble variants is the predominant (> 90%) transcript detected in eosinophilic HL-60C15 cells and in eosinophils obtained from cord blood cultures. Variable isoform mRNA expression has been observed in eosinophils purified from peripheral blood. The forced expiratory volume in 1 second in patients with mild asthma has been reported to be inversely correlated with the expression of the membrane-anchored isoform and directly correlated with the soluble isoform in eosinophils in endobronchial biopsies (Yasruel et al., 1997). In mouse B cells, transcripts encoding secreted variants are also generated through alternative splicing. In contrast to humans, there is no evidence for a similar soluble variant-specific exon. Rather, these soluble variant-specific transcripts are formed by skipping of the membrane anchor exon (Takaki et al., 1990; Tavernier et al., 1992; Imamura et al., 1994). The soluble hIL-5R isoform has antagonistic properties in vitro. It binds one IL-5 dimer in solution. It inhibits various IL-5 activities, including induced tyrosine phosphorylation of JAK2 and c, proliferation of IL-5-dependent cell lines, and eosinophilic differentiation and survival (Tavernier et al., 1991; Monahan et al., 1997), suggesting a role in the regulation of eosinophilia in vivo. No IL-5-potentiating effects have been observed in in vitro assays, underscoring its anti-inflammatory potential. So far, however, neither translation of the message encoding this soluble variant in vitro in eosinophils, nor circulating soluble hIL-5R in vivo has been reported. One possible explanation might be the thermolability of this soluble receptor. Alternatively, this splice regulation could merely serve a regulatory function driving transcription into a nonproductive pathway, reducing the expression level of the membraneassociated receptor. Soluble murine IL-5R also has antagonistic properties in vitro, albeit to a lesser degree than its human counterpart, consistent with its lower affinity for IL-5.

SIGNAL TRANSDUCTION Signal transduction via the IL-5 receptor involves ligand binding and receptor dimerization, requirements shared by other cytokine receptors (Bagley et al., 1997). The structural elements utilized by the IL-5 receptor  and chains to bind IL-5 have been discussed above. Following binding of IL-5, dimerization of the IL-5 receptor ensues, a process which shares certain events with the cytokine receptor superfamily at large but which has some features more limited to the IL-5, GM-CSF, and IL-3 subfamily of receptors. Dimerization of the IL-5 receptor involves the association of the IL-5 receptor  chain with c. This takes place by noncovalent as well as covalent means. The covalent linkage of IL-5R and c is probably the most functionally relevant one as it is associated with tyrosine phosphorylation of the receptor (Stomski et al., 1998). The cysteines involved are Cys86 and Cys91 in the most N-terminal domain of c (Stomski et al., 1998). These cysteines interact with a Cys in the IL-5 receptor  chain which has not yet been determined, however, since this  chain has an odd number of cysteines and all cysteines except Cys86 appear to form intramolecular bonds (based on alignment with other members of the cytokine receptor superfamily); Cys86 is the prime candidate. The related GM-CSF and IL-3 receptor  chains also exhibit an odd number of cysteines consistent with them also forming high-order complexes with c. Both the IL-5R and c subunits are required for signaling. Deletion of the cytoplasmic domains of either chain leads to complete loss of signaling, without altered ligand binding (Sakamaki et al., 1992; Takaki et al., 1994; Cornelis et al., 1995b). IL-5 binding leads to the rapid tyrosine phosphorylation of a multitude of cytoplasmic proteins. Mutations at position 13 of IL-5 cause diminished or abrogated activation of the c chain and may yield variants with antagonistic activity (Tavernier et al., 1995; Bagley et al., 1999). An E to K substitution at residue 13 leads to loss of detectable phosphorylation of c in eosinophils. Yet, whilst being deficient in inducing TF-1 proliferation and eosinophil adhesion, this IL-5 mutein still retains the capacity to support eosinophil survival, indicating that the different signaling pathways and functional responses can be segregated (McKinnon et al., 1997). The expression level of the IL-5R subunit may control this agonist/antagonist balance (van Ostade et al., 1999).

IL-5 Receptor 1907

Associated or intrinsic kinases Neither subunit of the IL-5 receptor possesses intrinsic tyrosine kinase activity. Studies in cell lines showed association of JAK2 and JAK1 (or JAK2) with the IL-5R and c subunits, respectively, and rapid tyrosine phosphorylation upon IL-5-binding (Ogata et al., 1998). This activation critically depends on the presence of intact, membrane-proximal proline-rich motifs (Box-1 motif ) in both chains (Quelle et al., 1994; Takaki et al., 1994).

Cytoplasmic signaling cascades In addition to the JAK kinases that associate directly with IL-5R, other kinases such as the Src family kinases Lyn and Fyn, and the Bruton tyrosine kinase, Btk, are activated by IL-5, suggesting a cascade of tyrosine phosphorylation events. Btk has been implicated in IL-5 signaling in B cells only. Mutations in the btk gene lead to B cell deficiencies in humans (X-linked agammaglobulinemia) and mice (X-linked immunodeficiency) (Hitoshi et al., 1993; Koike et al., 1995). Btk functions in concert with the Src family kinases Lyn and Fyn (Cheng et al., 1994; Appleby et al., 1995). Multiple tyrosine residues become phosphorylated on the c subunit and provide docking sites for signaling molecules. Both STAT1 (Pazdrak et al., 1995; van der Bruggen et al., 1995) and STAT5 (Mui et al., 1995) can become phosphorylated and activated by IL-5 treatment. STAT1 appears to be the major STAT activated by IL-5 in eosinophils. In the case of STAT5, a high degree of redundancy in recruitment sites has been reported (van Dijk et al., 1997). Via rapid recruitment and phosphorylation of the adapters Shc or SHP2, IL-5 signaling can be coupled to the Ras pathway (Pazdrak et al., 1997). Downstream effector molecules of Ras include PI-3 kinase and MAP kinase. PI-3 kinase plays a critical role in the induction by IL-5 of a chemokinetic response in bone marrow eosinophils (Palframan et al., 1998). The downstream targets of PI-3 kinase remain unclear. Activation of MAP kinase is required for induction of members of the AP-1 family, including the c-fos and c-jun proto-oncogenes. Members of this family are involved in myeloid differentiation (Foletta et al., 1998). Ras activation has also been implicated in suppression of apoptosis of eosinophils by IL-5. The c not only undergoes tyrosine phosphorylation but

is also phosphorylated on serine residues such as Ser585. This allows to bind to the 14-3-3 adapter protein and presumably associate with other molecules (Stomski et al., 1999). The biological significance of this is being determined.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated STAT1 (Pazdrak et al., 1995; van der Bruggen et al., 1995) and STAT5, c-fos, c-myc (Mui et al., 1995), NF-AT (Jinquan et al., 1999).

Genes induced Activation of STAT5 leads to rapid induction of immediate early response genes, including Cis, OSM, Id, pim-1, c-fos (Mui et al., 1995). Other genes induced include c-myc, VEGF (Horiuchi and Weller, 1997), 2 integrin (Palframan et al., 1998), PAF (Kishimoto et al., 1996), Bcl-xL (Dibbert et al., 1998) and Bcl2 (Ochiai et al., 1997; Dewson et al., 1999). The expression of the IL-3R, GM-CSFR, and c chains is induced, whereas that of the IL-5R is repressed (Wang et al., 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Although the IL-5 receptor exhibits the same biological activities as the GM-CSF and IL-3 receptors in a given cell type by virtue of their sharing the subunit, its unique biological effect stems from the spectrum of cells types that express the receptor. Thus, in contrast to the GM-CSF receptor and to a lesser degree the IL-3 receptor, which are more widely distributed, the expression of the IL-5 receptor is largely restricted to eosinophils and basophils in humans (and to B cells also in the mouse). Stimulation of the IL-5 receptor in vivo thus results in selective stimulation of eosinophil and basophil production which exacerbates allergic inflammation.

1908 Christopher J. Bagley, Jan Tavernier, Joanna M. Woodcock and Angel F. Lopez

Phenotypes of receptor knockouts and receptor overexpression mice Disruption of the IL-5R gene in mice leads to decreased levels of IgA in mucosal secretions (Hiroi et al., 1999) and an inability to induce eosinophilia in response to parasitic infection (Sugaya et al., 1997). Overexpression of the IL-5R does not lead to increased levels of eosinophils or B cells, although they exhibit some enhancement of sensitivity to IL-5 (Sugaya et al., 1997). Mice that are deficient in the chain of the IL-5 receptor are apparently normal except for a profound decrease in the number of eosinophils and a pulmonary alveolar proteinosis (PAP)-like disease (Nishinakamura et al., 1995). Given that the subunit is shared with the GM-CSF and IL-3 receptors, it is not clear which defect is specific for the IL-5 receptor. The reduction of eosinophils is likely to be so since a similar phenotype is seen in IL-5 knockouts; however, PAP is also seen in GM-CSF but not IL-5 knockout mice.

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain Soluble IL-5R inhibits the ability of IL-5 to promote the survival, proliferation, and activation of eosinophils and basophils.

Effects of inhibitors (antibodies) to receptors Inhibition of the IL-5 receptor is being tried as an alternative to inhibiting IL-5 itself for the treatment of allergic inflammation such as asthma. One approach relies on blocking the specific  chain of the IL-5 receptor by constructing IL-5 mutants defective in interacting with the chain only. This can be achieved by modifying IL-5 itself by mutating Glu13, a residue that is conserved in position in the tertiary structure and function in GM-CSF and IL-3. Substitution of Glu13 by a Gln, Arg, or Lys results in an IL-5 molecule that behaves as a specific IL-5 antagonist. However, the E13K mutant is still able to support eosinophil survival. A second approach involves blocking the subunit, an approach which has the additional therapeutical advantage of blocking stimulation by IL-5 but also the stimulation by

GM-CSF and IL-3, of eosinophils and basophils, the major inflammatory cell types in allergy. The antibody BION-1 has recently been developed and this binds to the B-C loop of the fourth domain of c and blocks the production, survival, and activation of eosinophils in response to IL-5, GM-CSF, and IL-3 (Sun et al., 1999).

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McKinnon, M., Page, K., Uings, I. J., Banks, M., Fattah, D., Proudfoot, A. E., Graber, P., Arod, C., Fish, R., Wells, T. N., and Solari, R. (1997). An interleukin 5 mutant distinguishes between two functional responses in human eosinophils. J. Exp. Med. 186, 121±129. Mita, S., Tominaga, A., Hitoshi, Y., Sakamoto, K., Honjo, T., Akagi, M., Kikuchi, Y., Yamaguchi, N., and Takatsu, K. (1989). Characterization of high-affinity receptors for interleukin 5 on interleukin 5-dependent cell lines. Proc. Natl Acad. Sci. USA 86, 2311±2315. Monahan, J., Siegel, N., Keith, R., Caparon, M., Christine, L., Compton, R., Cusik, S., Hirsch, J., Huynh, M., Devine, C., Polazzi, J., Rangwala, S., Tsai, B., and Portanova, J. (1997). Attenuation of IL-5-mediated signal transduction, eosinophil survival, and inflammatory mediator release by a soluble human IL-5 receptor. J. Immunol. 159, 4024±4034. Mui, A. L., Wakao, H., O'Farrell, A. M., Harada, N., and Miyajima, A. (1995). Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J. 14, 1166±1175. Murata, Y., Takaki, S., Migita, M., Kikuchi, Y., Tominaga, A., and Takatsu, K. (1992). Molecular cloning and expression of the human interleukin 5 receptor. J. Exp. Med. 175, 341±351. Nishinakamura, R., Nakayama, N., Hirabayashi, Y., Inoue, T., Aud, D., McNeil, T., Azuma, S., Yoshida, S., Toyoda, Y., Arai, K., Miyajima, A., and Murray, R. (1995). Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response, while beta IL3 receptordeficient mice are normal. Immunity 2, 211±222. Ochiai, K., Kagami, M., Matsumura, R., and Tomioka, H. (1997). IL-5 but not interferon-gamma (IFN-gamma) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression. Clin. Exp. Immunol. 107, 198±204. Ogata, N., Kouro, T., Yamada, A., Koike, M., Hanai, N., Ishikawa, T., and Takatsu, K. (1998). JAK2 and JAK1 constitutively associate with an interleukin-5 (IL-5) receptor a and bc subunit, respectively, and are activated upon IL-5 stimulation. Blood 91, 2264±2271. Pazdrak, K., Stafford, S., and Alam, R. (1995). The activation of the Jak-STAT 1 signaling pathway by IL-5 in eosinophils. J. Immunol. 155, 397±402. Pazdrak, K., Adachi, T., and Alam, R. (1997). Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival. J. Exp. Med. 186, 561±568. Palframan, R. T., Collins, P. D., Severs, N. J., Rothery, S., Williams, T. J., and Rankin, S. M. (1998). Mechanisms of acute eosinophil mobilization from the bone marrow stimulated by interleukin 5: the role of specific adhesion molecules and phosphatidylinositol 3-kinase. J. Exp. Med. 188, 1621±1632. Quelle, F. W., Sato, N., Witthuhn, B. A., Inhorn, R. C., Eder, M., Miyajima, A., Griffin, J. D., and Ihle, J. N. (1994). JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol. Cell. Biol. 14, 4335±4341. Sakamaki, K., Miyajima, I., Kitamura, T., and Miyajima, A. (1992). Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth, signal transduction and tyrosine phosphorylation. EMBO J. 11, 3541±3549. Stomski, F. C., Woodcock, J. M., Zacharakis, B., Bagley, C. J., Sun, Q., and Lopez, A. F. (1998). Identification of a Cys motif in the common beta chain of the interleukin 3,

1910 Christopher J. Bagley, Jan Tavernier, Joanna M. Woodcock and Angel F. Lopez granulocyte-macrophage colony-stimulating factor, and interleukin 5 receptors essential for disulfide-linked receptor heterodimerization and activation of all three receptors. J. Biol. Chem. 273, 1192±1199. Stomski, F. C., Dottore, M., Winnall, W., Guthridge, M. A., Woodcock, J., Bagley, C. J., Thomas, D. T., Andrews, R. K., Berndt, M. C., and Lopez, A. F. (1999). Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF. Blood 94, 1933±1942. Sugaya, H., Aoki, M., Yoshida, T., Takatsu, K., and Yoshimura, K. (1997). Eosinophilia and intracranial worm recovery in interleukin-5 transgenic and interleukin-5 receptor alpha chain-knockout mice infected with Angiostrongylus cantonensis. Parasitol. Res. 83, 583±590. Sun, Z., Yergeau, D. A., Tuypens, T., Tavernier, J., Paul, C. C., Baumann, M. A., Tenen, D. G., and Ackerman, S. J. (1995). Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor alpha subunit gene. J. Biol. Chem. 270, 1462±1471. Sun, Q., Jones, K., McClure, B., Cambareri, B., Zacharakis, B., Iversen, P. O., Stomski, F., Woodcock, J. M., Bagley, C. J., D'Andrea, R., and Lopez, A. F. (1999). Simultaneous antagonism of IL-5, GM-CSF and IL-3 stimulation of human eosinophils by targetting the common cytokine binding site of their receptors. Blood 94, 1943±1951. Takaki, S., Tominaga, A., Hitoshi, Y., Mita, S., Sonoda, E., Yamaguchi, N., and Takatsu, K. (1990). Molecular cloning and expression of the murine interleukin-5 receptor. EMBO J. 9, 4367±4374. Takaki, S., Mita, S., Kitamura, T., Yonehara, S., Yamaguchi, N., Tominaga, A., Miyajima, A., and Takatsu, K. (1991). Identification of the second subunit of the murine interleukin5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor. EMBO J. 10, 2833±2838. Takaki, S., Kanazawa, H., Shiiba, M., and Takatsu, K. (1994). A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction. Mol. Cell. Biol. 14, 7404±7413. Tavernier, J., Devos, R., Cornelis, S., Tuypens, T., Van der Heyden, J., Fiers, W., and Plaetinck, G. (1991). A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific alpha chain and a beta chain shared with the receptor for GM-CSF. Cell 66, 1175±1184. Tavernier, J., Tuypens, T., Plaetinck, G., Verhee, A., Fiers, W., and Devos, R. (1992). Molecular basis of the membraneanchored and two soluble isoforms of the human interleukin 5 receptor alpha subunit. Proc. Natl Acad. Sci. USA 89, 7041± 7045.

Tavernier, J., Tuypens, T., Verhee, A., Plaetinck, G., Devos, R., Van der Heyden, J., Guisez, Y., and Oefner, C. (1995). Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist. Proc. Natl Acad. Sci. USA 92, 5194±5198. Tuypens, T., Plaetinck, G., Baker, E., Sutherland, G., Brusselle, G., Fiers, W., Devos, R., and Tavernier, J. (1992). Organization and chromosomal localization of the human interleukin 5 receptor alpha-chain gene. Eur. Cytokine Netw. 3, 451±459. van der Bruggen, T., Caldenhoven, E., Kanters, D., Coffer, P., Raaijmakers, J. A., Lammers, J. W., and Koenderman, L. (1995). Interleukin-5 signaling in human eosinophils involves JAK2 tyrosine kinase and Stat1 alpha. Blood 85, 1442±1448. van Dijk, T. B., Caldenhoven, E., Raaijmakers, J. A., Lammers, J. W., Koenderman, L., and de Groot, R. P. (1997). Multiple tyrosine residues in the intracellular domain of the common beta subunit of the interleukin 5 receptor are involved in activation of STAT5. FEBS Lett. 412, 161±164. van Ostade, X., Van der Heyden, J., Verhee, A., Vandekerckhove, J., and Tavernier, J. (1999). The cell surface expression level of the human interleukin-5 receptor alpha subunit determines the agonistic/antagonistic balance of the human interleukin-5 E13Q mutein. Eur. J. Biochem. 259, 954±960. Wang, P., Wu, P., Cheewatrakoolpong, B., Myers, J. G., Egan, R. W., and Billah, M. M. (1998). Selective inhibition of IL-5 receptor alpha-chain gene transcription by IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor in human blood eosinophils. J. Immunol. 160, 4427±4432. Woodcock, J. M., Zacharakis, B., Plaetinck, G., Bagley, C. J., Sun, Q., Hercus, T. R., Tavernier, J., and Lopez, A. F. (1994). Three residues in the common chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF. EMBO J. 13, 5176±5185. Woodcock, J. M., Zacharakis, B., Plaetinck, G., Bagley, C. J., Sun, Q., Hercus, T. R., Tavernier, J., and Lopez, A. F. (1996). A single tyrosine residue in the membrane proximal domain of the GM-CSF, IL-3 and IL-5 receptor common chain is necessary and sufficient for high affinity binding and signalling by all three ligands. J. Biol. Chem. 271, 25999±26006. Yasruel, Z., Humbert, M., Kotsimbos, T. C., Ploysongsang, Y., Minshall, E., Durham, S., Pfister, R., Menz, G., Tavernier, J., Kay, A. B., and Hamid, Q. (1997). Membrane-bound and soluble alpha IL-5 receptor mRNA in the bronchial mucosa of atopic and nonatopic asthmatics. Am. J. Respir. Crit. Care Med. 155, 1413±1418. Zhang, J., Kuvelkar, R., Cheewatrakoolpong, B., Williams, S., Egan, R. W., and Billah, M. M. (1997). Evidence for multiple promoters of the human IL-5 receptor alpha subunit gene: a novel 6-base pair element determines cell-specific promoter function. J. Immunol. 159, 5412±5421.

IL-5 Receptor 1911

LICENSED PRODUCTS See Table 2.

Table 2 Suppliers of anti-IL-5R and anti-IL-5R antibodies Type

Class Clone/ID

Supplier

Anti-IL-5R antibodies Polyclonal (goat)

IgG

AF-253-NA R&D Systems

Monoclonal

IgG1 A14

Pharmingen

Monoclonal

IgG1 MAB1008

Chemicon International

Monoclonal

IgG1 202325

Stratagene

Monoclonal

IgG2b S16

Santa Cruz

Monoclonal

IgG1k AR-1635

Maine Biotechnology Services

Monoclonal

IgG1 3D7

Pharmingen

Monoclonal

IgG1 4F3

Amrad

Anti-IL-5R antibodies