Characterization of cDNAs encoding the murine interleukin 2 receptor ( IL-2 R ) y chain : Chromosomal mapping and tissue specificity of IL-2 R y chain expression

The IL-2R y chain (IL-MRy) is an essential component of highand intermediate-affinity IL-2Rs, playing critical roles for ligand binding and internalization. Recently, our laboratory has demonstrated that IL-2R'y mutation results inX chromosome-linked severe combined immunodeficiency in humans, suggesting that IL-2Ry plays a vital role in thymic maturation of human T cells. We now report the isolation and characterization ofcDNAs encoding murine IL-2Ry. The open reading frame encodes 369 aa, identical in length to that encoded by the human IL-2Ry cDNA. Murine IL-2Ry and human IL-2Ry have 69% and 70% identity at the nudeotide and amino acid levels, respectively. As expected, the murine IL-2Ry retains the WSXWS motif and four cysteine residues characteristic of cytokine receptor superfamily members. IL2Ry mRNA distribution shows significant tissue specificity, with particularly high-level expression in spleen and thymus, and higher expression in single-positive (CD4+8or CD4-8+)enriched thymocytes than in double-negative (CD4-8-) thymocytes. Finafly, we have localized the murine IL-2Ry gene, 1I2rg, to the X chromosome between Rsvp and Plp and demonstrated that a defect in IL-2Ry is not responsible for the X chromosome-linked xid mutation, which maps to this same region. The cloning of the murine IL-Ry cDNA will facilitate the investigation of the role of this protein in lymphocyte function and thymic development. Interleukin 2 (IL-2) and IL-2Rs critically regulate the magnitude and duration of the T-cell immune response after antigen activation (for review, see refs. 1-3). Three classes of IL-2Rs exist. Resting lymphocytes express intermediateaffinity IL-2Rs, whereas activated lymphocytes express highand low-affinity IL-2Rs (1-3). The highand intermediate-affinity IL-2Rs can transduce IL-2 signals, but the low-affimity IL-2Rs cannot (1-3). Different combinations of three chains, denoted the a (4, 5), p (6-12), and 'y (13-17) chains, form these three classes of IL-2Rs. Low-affnity IL-2Rs contain IL-2Ra, but not IL-2R,B or IL-2Ry; intermediate-affinity IL-2Rs contain IL-2R( and IL-2Ry chains, but not IL-2Ra; and high-affinity IL-2Rs contain all three chains. Thus, IL-2R,B and IL-2Ry are components of high and intermediate IL-2Rs, the receptors capable of transducing IL-2-mediated proliferative and cytolytic signals. IL-2R,B has been extensively characterized, but studies of IL-2Ry are more limited. Human IL-2Ra (4,5), IL-2R,B(12), and IL-2Ry(15) cDNAs have been isolated, but murine cDNAs have only been identified for IL-2Ra (18,19) and IL-2RP (20). We now report the cloning of the murine IL-2Ry cDNA, its chromosomal The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. mapping, and characterization of IL-2Ry mRNA expression in different tissues. § This represents an important step to facilitating the study of the functional and developmental roles of IL-2Ry. Such studies are particularly important in view of recent data indicating that mutations of IL-2Ry in humans result in X chromosome-linked severe combined immunodeficiency (XSCID) (21), a disease characterized by absent or profoundly diminished T cells and severely depressed cell-mediated and humoral immunity (for review, see refs. 22 and 23). MATERIAL AND METHODS Oligonucleotides and DNA Sequence Analysis. Oligonucleotides were synthesized on an Applied Biosystems model 392 DNA/RNA synthesizer and are summarized in Table 1. All DNA sequencing was performed by the dideoxynucleotide chain-termination method, using Sequenase Version 2.0 (United States Biochemical). Hybridization Probe and PCR Conditions. Primers y3 and 'ylO corresponding to the human IL-2Ry sequence (see Table 1) were used to amplify ==2 ,g ofDNA derived from a cDNA library prepared from murine 32D myeloid progenitor cells (24). These cells are capable of responding to IL-2 after transfection of IL-2R(3 (25) and, therefore, were assumed to express endogenous murine IL-2Ry. PCR amplification was performed with 30 cycles of 94°C, 50°C, and 72°C, each for 1 min. The resulting 660-bp fragment was isolated and designated as the y3-10 PCR probe. Identification of a Full-Length IL-2Ry cDNA. A cDNA library in AgtlO prepared from mRNA from Con A-activated murine splenocytes (Clontech, 3 x 105 clones) was initially screened with the y3-10 PCR probe labeled with 32P by nick translation. Duplicate nitrocellulose filters were hybridized in Nitrohyb (Digene Diagnostics, Silver Spring, MD) solution overnight at 42°C and washed under high-stringency conditions (twice in 2x SSPE/0.1% SDS at room temperature for 15 min and once in 0.lx SSPE/0.1% SDS at 56°C for 30 min; lx SSPE = 0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA). The insert from one clone, denoted 9E, was subcloned into pBluescript II SK(+) and sequenced using Sp6 and T7 primers. The insert was 798 bp long and had 60%6 sequence identity to the human cDNA. This partial-length cDNA was used to screen another murine cDNA library in the BstXI site of pcDNAI (Invitrogen) prepared from Con A-activated splenocytes from CBA/Ca mice. Five clones were identified, and combination of two of these clones yielded the full-length cDNA sequence. Initial sequencing Abbreviations: IL-2, interleukin 2; IL-2R, IL-2 receptor; XSCID, X chromosome-linked severe combined immunodeficiency. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. L20048).