Biochemical Identity and Characterization of the Mouse Interleukin-2 Receptor β and γ c Subunits

Journal of Biological Chemistry

2000

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The common ␥ chain (␥c), a subunit of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors, contributes to both cytokine binding and subsequent signal transduction. Using a model-based site-directed mutagenesis strategy, we have identified residues of the mouse ␥c extracellular domain that are required for normal ␥c-dependent enhancement of IL-2 and IL-7 binding. One of these sites, Tyr-103, is homologous to key ligand-interacting residues in the growth hormone and erythropoietin receptors, whereas Cys-161, Cys-210, and Gly-211 may function indirectly by maintaining the functional conformation of ␥c via formation of an intramolecular disulfide bond. These two cysteines are also required for the integrity of a putative epitope recognized by TUGm2, an antagonistic monoclonal antibody that blocks ␥c-dependent cytokine binding and bioactivity. These results are consistent with the involvement of three predicted loops in ␥c that contribute to the binding of both IL-2 and IL-7. Mutations in these loops have also been noted in the ␥c gene of patients with X-linked severe combined immunodeficiency.The common ␥ chain (␥c) 1 is a member of the type I cytokine receptor superfamily and functions as a shared subunit of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (reviewed in Refs. 1 and 2). In each of these receptors ␥c regulates signaling via the JAK3 tyrosine kinase associated with its cytoplasmic tail. The biological importance of ␥c is most dramatically illustrated by the fact that mutations of either ␥c or JAK3 are the primary causes of human X-linked severe combined immunodeficiency (X-SCID), characterized by a failure in T and natural killer cell development (3). Although ␥c does not detectably bind cytokines by itself, its recruitment into the IL-2, IL-4, and IL-7 receptor complexes enhances ligand binding. This is most evident in the context of the IL-2 receptor (IL-2R) where ␤␥ heterodimers bind IL-2 with 100-fold higher affinity than IL-2R␤ (4), and the affinity of ␣␤␥ trimers is 10-fold increased over ␣␤ complexes (5). A similar but more modest 3-5-fold increase in affinity has also been observed in the IL-4R (6) and IL-7R (7).Cytokines binding to type I cytokine receptors are typically folded as bundles of four ␣-helices. The receptor subunits, including ␥c, are characterized by extracellular homology regions composed of two fibronectin type III domains that are each composed of seven ␤-strands. The first domain contains four highly conserved cysteine residues linked by disulfide bonds whereas the second domain contains a highly conserved WSXWS motif (8). The prototypical members of the type I cytokine receptor superfamily are the growth hormone and erythropoietin receptors (EPORs) for which the crystal structure has been determined for the extracellular portion of the receptors together with their ligands (9, 10). The structure of the growth hormone receptor (GHR) complex (9) has been used as a template for homology modeling of a number of receptors within the family. Subsequent use of such m...

Section: Resultsmentioning

confidence: 88%

Three Loops of the Common γ Chain Ectodomain Required for the Binding of Interleukin-2 and Interleukin-7

Olosz

Journal of Biological Chemistry

2000

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“…FITCanti-CD4 (GK1.5), biotin-anti-IL-2R␣ (7D4), biotin-anti-B220, anti-IL-2 (S4B6) (15), anti-␥c (4G3 and 3E12) (16), and anti-IL-2R␤ (5H4) (17) were prepared in our laboratory. FACS analysis was performed as previously described (18) using a Becton Dickinson FACScan and CellQuest software (Mountain View, CA).…”

Section: Abs and Facs Analysismentioning

confidence: 99%

Normal Lymphoid Homeostasis and Lack of Lethal Autoimmunity in Mice Containing Mature T Cells with Severely Impaired IL-2 Receptors

The Journal of Immunology

Porter

Codias

et al. 2000

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139

137

The importance of IL-2Rβ function for immune regulation is highlighted by the severe impairment in lymphoid cell function in IL-2Rβ-deficient mice. It has been speculated that failed IL-2/IL-2R signaling in peripheral T cells causes the associated autoimmunity, imbalanced peripheral lymphoid homeostasis, and defective T cell function. This study explored the requirement for IL-2Rβ function in mature T lymphocytes. We show that transgenic thymic expression of the IL-2R β-chain in IL-2Rβ-deficient mice prevents lethal autoimmunity, restores normal production of B lymphocytes, and results in a peripheral T cell compartment that is responsive to triggering through the TCR, but not the IL-2R. The dysfunction of the IL-2R is illustrated by the near complete failure of mature T cells to proliferate to IL-2 in vitro and in vivo, to differentiate into CTL, and to up-regulate IL-2Rα expression. These data indicate that lymphoid homeostasis is largely maintained despite a nonfunctional IL-2R in mature T lymphocytes and suggest that IL-2Rβ provides an essential signal during thymic development to regulate self-reactivity.

“…Polyclonal rabbit anti-mouse bax (P-19) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Purified mAbs specific for IL-2R␤ (5H4) (26) and IL-7R␣ (A7R34; kindly provided by S. Nishikawa, Kyoto University, Kyoto, Japan) (27) have been described previously. For IL-7-binding studies, binding medium consisted of RPMI 1640 with 2.5% FCS, 15 mM HEPES, and sodium azide (1 mg/ml).…”

Section: Reagents and Absmentioning

confidence: 99%

Control of T Cell Development In Vivo by Subdomains Within the IL-7 Receptor α-Chain Cytoplasmic Tail

Porter

Scibelli

The Journal of Immunology

2001

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IL-7/IL-7R signaling functions in both growth and differentiation during T cell development. In this study, we examined the extent these activities were controlled by signaling associated with distinct IL-7Rα cytoplasmic domains by transgenic expression of wild-type or cytoplasmic deletion mutants of IL-7Rα in the thymi of IL-7Rα−/− mice. We show an essential requirement for the tyrosine-containing carboxyl-terminal T domain in restoring thymic cellularity, pro-/pre-T cell progression, and survival. In contrast, the functional differentiation of TCR αβ cells and the development of TCR γδ cells are partially independent of the T domain. Thus, separate cytoplasmic domains of the IL-7Rα chain differentially control distinct functions during T cell development, whereas normal IL-7R-dependent thymic development requires the integrated activity of all these domains.