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

Abstract

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...

Crystal structure of the IL-2 signaling complex: Paradigm for a heterotrimeric cytokine receptor

“…Most strikingly, little specificity is conferred upon the IL-2͞␥ c interface due to the formation of only 2 hydrogen bonds, as opposed to 7 and 10 in the other two IL-2͞IL-2R interfaces. Our structural results also agree well with extensive mutagenesis work on defining the epitopes on ␥ c for various cytokines (25,26,35). Tyr-103 ␥ , Cys-160 ␥ , and Cys-209 ␥ , identified as hot-spot residues in all of the cytokine͞␥ c interfaces, form a hydrophobic patch in the center of the IL-2͞␥ c interface.…”

“…Tyr-103 ␥ , His-159 ␥ , and Leu-208 ␥ together with the disulfide (Cys-160 ␥ -Cys-209 ␥ ) contribute 53% of the buried surface on ␥ c and represent the major binding determinant. The importance of the disulfide bridge was previously appreciated from mutagenesis data (25,26), but its direct involvement in ligand binding was unexpected. With a buried surface area of 72 Å 2 and one hydrogen bond, IL2 is the most critical IL-2 residue that contacts ␥ c in accordance with biochemical data (27).…”

Identification of Ligand-binding Site III on the Immunoglobulin-like Domain of the Granulocyte Colony-stimulating Factor Receptor

“…The available data about sites of interaction of cytokine receptors with their specific ligands indicated that the loop regions of FNIII domains rather than ␤ strands were the most likely ligand-binding sites (32)(33)(34)(35)(36). An alignment of Ig-like domains was used to predict the ␤ strands and loop regions of the G-CSF-R Ig-like domain.…”

Structural Basis for Binding Multiple Ligands by the Common Cytokine Receptor γ-Chain

“…The pDC302 expression vector containing the full-length mouse IL-4R␣ (mIL-4R␣) cDNA was described previously (22) (provided by Immunex Corp., Seattle, WA). Site-directed mutagenesis of the m␥ c gene was performed by the QuikChange method (Stratagene, La Jolla, CA), as previously described (17). Mutations were verified by DNA sequencing.…”

“…Recent structure-function analysis of the mouse ␥ c (m␥ c ) ectodomain by site-directed mutagenesis (17), guided by predictions of theoretical models of the ␥ c structure (18 -20), identified four amino acids of ␥ c that are necessary for binding IL-2 and IL-7 (17). One of these residues, Tyr-103, may be directly involved in IL-2 and IL-7 binding.…”