IL-9 Receptor

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IL-9 Receptor Jean-Christophe Renauld* Ludwig Institute for Cancer Research, Catholic University of Louvain, 74 Avenue Hippocrate, Brussels, B-1200, Belgium * corresponding author tel: 32-2-764-7464, fax: 32-2-762-9405, e-mail: [email protected] DOI: 10.1006/rwcy.2000.14004.

SUMMARY High-affinity binding of IL-9 to target cells is mediated by a heterodimer consisting of the IL-9 receptor  chain and the IL-2 receptor chain, also called c (common ); both chains belong to the hematopoietin receptor superfamily. The IL-9 receptor  chain is sufficient to confer high-affinity binding but both chains are needed for signal transduction. The IL-9 receptor  is associated with JAK1 tyrosine kinase, and the IL-2 receptor chain with JAK3. Upon IL-9 binding, both kinases are activated and the IL-9 receptor is phosphorylated on a single tyrosine. This phosphorylated residue is used as a docking site for STAT1, STAT3, and STAT5 transcription factors. Activation of these transcription factors is considered to be critical for all IL-9 activities, because a mutant on this single tyrosine residue completely lost its activity. The human IL-9R gene is located on the long arm pseudoautosomal region of the X and Y chromosomes, a region which has been linked to asthma.

BACKGROUND

Discovery The mouse IL-9R cDNA was identified by expression cloning in COS cells (Renauld et al., 1992). The human IL-9 receptor cDNA was isolated by crosshybridization with a mouse probe (Renauld et al., 1992).

Structure The IL-9 receptor (IL-9R) was found to interact with the chain of the IL-2R, which is required for signal

transduction but not for IL-9 binding, since an antibody directed against this molecule completely inhibits the activity of IL-9 without affecting the Kd of IL-9 binding (Kimura et al., 1995). The fact that this molecule, now called c, is shared by IL-2R, IL-4R, IL7R, IL-9R, and IL-15R could explain the overlapping activities observed for these T cell growth factors.

Main activities and pathophysiological roles The IL-9R is believed to mediate all the biological activities of IL-9 on T cells, B cells, mast cells, eosinophils, and hematopoietic progenitors, particularly for the development of asthma, for which a genetic linkage has been reported (Holroyd et al., 1998).

GENE The human genome contains at least four IL-9R pseudogenes with  90% homology with the IL-9R gene (Kermouni et al., 1995; Vermeesch et al., 1997).

Accession numbers Human IL-9R gene: L39064 Human IL-9R pseudogenes: L39063, L39062

Sequence The mouse IL-9R gene is composed of nine exons and eight introns, sharing many characteristics with other genes encoding cytokine receptors (Renauld et al., unpublished data). The human IL-9R gene is composed of 11 exons and 10 introns, stretching over  17 kb.

1492 Jean-Christophe Renauld A frequent alternative splicing of the human gene generates an intriguing heterogeneity in the 50 untranslated region of the mRNA and introduces some short open reading frames that might represent an additional level in the regulation of IL-9R translation, as suggested for many genes involved in cell growth (Kozak, 1991; Kermouni et al., 1995). More recently, another splice variant was identified that contained an in-frame deletion of a single residue of the extracellular domain and lacked the ability to bind IL-9 (Grasso et al., 1998). Analysis of the 50 flanking region revealed multiple transcription initiation sites as well as potential binding motifs for AP-1, AP-2, AP-3, SP-1, and NFB, although this region lacks a TATA box (Kermouni et al., 1995). Although the IL-9R pseudogenes are similar to the IL-9R gene ( 90% identity), none of these copies encodes a functional receptor: none of them contain sequences homologous to the 50 flanking region or exon 1 of the IL-9R gene and the remaining open reading frames have been inactivated by various point mutations and deletions (Kermouni et al., 1995).

Chromosome location and linkages In the mouse, the IL-9R gene is a single copy gene located on chromosome 11 (Vermeesch et al., 1997). The human IL-9R gene is located in the subtelomeric region of chromosomes X and Y. IL-9R was thus the first gene to be identified in the long arm pseudoautosomal region and turned out to be a unique tool to study this particular region of the genome. Using a polymorphism in the coding region of this gene, Vermeesch and colleagues showed that IL-9R is expressed both from X and Y and escapes X inactivation (Vermeesch et al., 1997). Interestingly, a genetic linkage has been reported between this region and asthma or bronchial hyperresponsiveness, suggesting that different alleles of the IL-9R gene affect allergic responses (Holroyd et al., 1998). The four IL-9R pseudogenes are located in the subtelomeric regions of chromosomes 9q, 10p, 16p, and 18p (Kermouni et al., 1995).

PROTEIN

Accession numbers SwissProt: Mouse: Q01114 Human: Q01113

Description of protein The murine IL-9R contains 468 amino acids, including an extracellular domain, composed of 233 amino acids (Renauld et al., 1992) that shows the typical features of the hematopoietin receptor superfamily, namely four conserved cysteines and a WSEWS motif, located a few residues upstream from the transmembrane domain (Bazan, 1990). The human IL-9R cDNA encodes a 522 amino acid protein.

Relevant homologies and species differences The human IL-9R protein shows a 53% identity with the mouse IL-9R. The extracellular region is particularly conserved with 67% identity, while the cytoplasmic domain is significantly larger in the human receptor (231 versus 177 residues) (Renauld et al., 1992). The juxtamembrane region of the cytoplasmic tail of the IL-9R contains a Pro-X-Pro sequence preceded by a cluster of hydrophobic residues, which partially fits a consensus motif shared by many cytokine receptors (IL-4R, IL-7R, IL-3R, EPOR, IL-2R , G-CSFR) (Murakami et al., 1991). Downstream from this Pro-X-Pro motif, a striking homology was observed with the chain of the IL-2R and with the erythropoietin receptor. As a result, for the first 33 amino acids of the cytoplasmic domain, a 40% identity was noticed between the human IL-9R and the IL-2R . This homologous region probably explains why IL-9, like other cytokines such as IL-2, induces JAK1 and JAK3 phosphorylation (Russell et al., 1994; Yin et al., 1994; Demoulin et al., 1996).

Affinity for ligand(s) A variety of mouse hematopoietic cells express highaffinity receptors for IL-9 (Kd  100 pM) (Druez et al., 1990). The chain of the IL-2R, which associates with the IL-9R, is required for signal transduction but not for IL-9 high-affinity binding (Kimura et al., 1995).

Cell types and tissues expressing the receptor This issue has been poorly investigated so far. A variety of mouse hematopoietic cells, including T cells, mast cells, and macrophages, express high-affinity receptors for IL-9 (Druez et al., 1990). More recently,

IL-9 Receptor 1493 IL-9R was found to be expressed preferentially by peritoneal B-1 lymphocytes.

Release of soluble receptors Soluble receptors have never been reported at the protein level. As observed for many members of the hematopoietin receptor superfamily, IL-9R mRNA has been identified that lack the sequences encoding the transmembrane and cytoplasmic domains, as a result of alternative splicing (Renauld et al., 1992). However, the frequency of these mRNA seems quite low and it is not yet clear whether they really encode a soluble IL-9-binding protein.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases IL-9R interacts with the chain of the IL-2R, which is required for signal transduction and is shared by IL-2R, IL-4R, IL-7R, IL-9R, and IL-15R (Demoulin and Renauld, 1998). So far, the only function of c seems to recruit the tyrosine kinase JAK3, while IL9R is associated with JAK1. Upon IL-9 binding, both JAK1 and JAK3 become phosphorylated and catalytically active. These kinases are likely to be responsible for IL-9R phosphorylation on one of its five tyrosine residues.

JAK1 activation require the same region of the IL-9R (Demoulin et al., unpublished data) and these two molecules were shown to be associated in response to IL-9 (Yin et al., 1995). Taken together, these observations raise the possibility that, upon IL-9 activation, 4PS/IRS2 becomes phosphorylated by interacting directly with the JAK1 tyrosine kinase. After phosphorylation, 4PS/IRS2 binds the SH2 domain of various signaling proteins including the p85 subunit of the phosphatidylinositol 3-kinase (Demoulin et al., unpublished data).

DOWNSTREAM GENE ACTIVATION

Transcription factors activated STAT1, STAT3, and STAT5 (Demoulin et al., 1996).

Genes induced Granzyme A, granzyme B, mouse mast cell proteases (MMCP), Ly6A/E, L-selectin, IL-6, adseverin (Robbens et al., 1998), Bcl-3 (Richard et al., 1999), M-ras (Louahed et al., 1999).

Promoter regions involved Cytoplasmic signaling cascades This single phosphorylated residue acts as a docking site for STAT1, STAT3, and STAT5 ± three transcription factors that, after phosphorylation by the JAK kinases associated to the receptor, form heteroor homodimers and migrate to the nucleus (Demoulin et al., 1996; Bauer et al., 1998). IL-9 does not seem to induce or enhance the phosphorylation of the serine/threonine kinases Raf-1 or MAP kinases in the Mo7E leukemia cell line (Miyazawa et al., 1992), or in mouse lymphoid cells (Grasso et al., unpublished). More clearly established is the activation by IL-9 of an adaptor protein called 4PS/IRS2, a feature shared with IL-4 signal transduction, where this pathway was shown to be critical for growth regulation (Keegan et al., 1994; Yin et al., 1995; Demoulin et al., 1996). Phosphorylation of 4PS/IRS2 is not dependent on the phosphorylation of the IL-9R, contrasting with the IL-4 system in which 4PS/IRS2 associates with the IL-4R through a phosphotyrosine residue. Preliminary observations suggest that 4PS/IRS2 and

GAS site for Ly6A/E (Demoulin et al., 1999).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors See chapter on IL-9 activities.

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