Expression cloning of the murine and human interleukin 9 receptor cDNAs.

Abstract

Interleukin 9 (IL-9) is a T-cell-derived lymphokine that induces the proliferation of various lymphold and hemopoietic cells. A cDNA done encoding the murine receptor was isolated by expression cloning in COS cells and screening with 12SI-labeled IL-9. Transient expression of this cDNA produced high-affinity binding sites for IL-9. The predcid 52-kDa protein contains a putative signal peptide and a pical trsembrane domain. A cDNA for the human homoogue was isolated by cross-hybridization. Transfection of this cDNA in a murine T-cell cone conferred responsiveness to human IL-9. Sequence analysis revealed that the IL-9 receptor belongs to the recently described hematopoletin receptor superfamily and is expressed in membrane-bound and soluble forms.Interleukin 9 (IL-9) was originally identified as a growth factor for murine T helper cell clones (1, 2), murine mast cell lines (3, 4), and a human megakaryoblastic leukemia line (5). Subsequently, additional biological targets were discovered including murine fetal thymocytes (6), murine erythroid progenitors (7), human T-cell lines (8), and human erythroid and myeloid precursors (9, 10). Moreover, involvement of IL-9 in tumorigenesis has been recently suggested by the observations that a murine T helper clone becomes tumorigenic after transfection with the IL-9 cDNA (11) and that lymph nodes from patients with Hodgkin disease or large-cell anaplastic lymphoma express IL-9 constitutively (12). Preliminary characterization of the IL-9 receptor on a murine T-cell clone has demonstrated the existence of saturable and specific binding sites with a Kd of -100 pM. Cross-linking analysis showed that the IL-9 receptor consists of a 64-kDa glycoprotein, the molecular mass of which is reduced to 54 kDa on treatment with N-glycosidase F (13).We report here the expression cloning and sequence of a cDNA encoding the murine IL-9 receptor.t This cDNA was further used to identify a human homologue.t MATERIALS AND METHODS Construction of cDNA Libraries. Poly(A)+ RNA extracted from the murine T-cell clone TS1 (1) was converted to double-stranded cDNA by the method of Gubler and Hoffman (14) with random hexanucleotide primers. EcoRI adaptors were attached and cDNA larger than 1.5 kilobases (kb) was isolated by fractionation on a 5-201% (wt/vol) potassium acetate gradient (15). The size-selected cDNA was inserted into the EcoRI site of the pCDSRa expression vector (16). Two additional cDNA libraries were generated in the BstXI site of the pCDM8 vector (15) with oligo(dT) or random primers. A human cDNA library was constructed by the same method in the pRC/RSV plasmid (Invitrogen, San Diego) with RNA from the megakaryoblastic leukemia cell line Mo7E (17).Transient Expression and Biding Assays. Purified recombinant murine IL-9 was radiolabeled using the Bolton and Hunter reagent as described (18) to yield material with a specific activity of 2300 cpm/fmol. Screening of cDNA pools by direct expression in COS cells was performed essentially as described by Gearing et al. (19). The mu...

Cytokines and effector T cell subsets causing autoimmune CNS disease

“…Minor amounts of IL-9 may also be produced by Th2 and Th17 cells. IL-9 activates a heterodimeric receptor consisting of the cytokinespecific IL-9 receptor a chain (IL-9Ra) and the common c-chain [91]. Upon binding of IL-9 to its receptor, the kinases Jak1 and Jak3 are phosphorylated and subsequently activate Stat1, Stat3 and Stat5 [92][93][94].…”

Interleukin‐6: Structure‐function relationships

“…It was recently shown that two of these latter residues are also important for activation of STAT1 (consensus sequence Tyr-Xaa-Pro-Gln) (Gerhartz et al, 1996). The box 3 motif is also found in the receptors for IL-7 (Goodwin et al, 1990), IL-9 (Renauld et al, 1992), IL-10 (Liu et al, 1994), and thrombopoietin (Vigon et al, 1992), in the OSMspecific receptor (OSMRP) (Mosley et al, 1996), and in the leptin receptor (OB-R) (Tartaglia et al, 1995). The OSMRP and the OB-R are related to gp130 in primary structure (23.3% and 20.7% overall amino acid identity, respectively) and in modular structure, featuring both Ig-like and FN III domains (Hibi et al, 1990;Tartaglia et al, 1995;Mosley et al, 1996).…”

Soluble IL7Rα potentiates IL-7 bioactivity and promotes autoimmunity

“…Soluble IL7Rα (sIL7Rα) was identified in 1990 coincident with cloning of human cell-associated IL7Rα, and sIL7Rα is known to circulate in nanomolar concentrations (21)(22)(23)(24), but, to date, the biological function of sIL7Rα remains unclear. There are two primary mechanisms through which soluble cytokine receptors can be produced (17): shedding of membrane-bound receptors [e.g., TNF receptor 2 (25)] and alternative splicing leading to a protein lacking the transmembrane domain [e.g., IL-9Rα (26)]. Previous biochemical studies have demonstrated that sIL7Rα present in human plasma is primarily derived from alternative splicing and comprises an isoform lacking exon 6 (Δ6IL7Rα), as well as a unique 26-aa sequence as a result of a frame shift and a premature stop codon (24) (Fig.…”