Oligomerization of IL-2Ralpha

Interleukin (IL) 2 receptor subunit alpha (IL-2Ralpha) increases the affinity of the IL-2 receptor complex while hetero-association of IL-2Rbeta and gamma(c) chains initiates a proliferative signal. We show here that IL-2Ralpha is necessary for receptor clustering required for augmentation of IL-2 signalling. Cells expressing chimeras incorporating the extracellular domain of IL-2Ralpha demonstrated IL-2 independent homo-association of the IL-2Ralpha chimera. Singly or co-transfected IL-2Rbeta and gamma(c) chimeras showed no spontaneous or IL-2-inducible oligomerization. Co-transfection of IL-2Ralpha and IL-2Rbeta (+/- gamma(c)) chimeras diminished spontaneous IL-2Ralpha chimera oligomerization and permitted IL-2-inducible hetero-oligomerization of receptor components. Homo-association of IL-2Ralpha was also demonstrated by fluorescence resonance energy transfer (FRET). The spontaneous homo-oligomerization property of IL-2Ralpha required the membrane proximal region of the receptor (exon 6) by deletion analysis; the IL-2 inducible oligomerization property of IL-2Ralpha required the second "sushi" domain (exon 4). This work provides insight into the mechanics of this complex receptor system and to other receptor complexes in the immune system that send signals by clustering receptor subunits.     

Identification of distinct roles for a dileucine and a tyrosine internalization motif in the interleukin (IL)-13 binding component IL-13 receptor alpha 2 chain

Interleukin (IL)-13 receptor alpha2 (IL-13Ralpha2) chain is an essential binding component for IL-13-mediated ligand binding. Recently, we have demonstrated that this receptor chain also plays an important role in the internalization of IL-13. To study the mechanism of IL-13 internalization, we generated mutated IL-13Ralpha2 chains that targeted trileucine residues (Leu(335), Leu(336), and Leu(337)) in the transmembrane domain and a tyrosine motif (Tyr(343)) in the intracellular domain and transfected these cDNAs in COS-7 cells. Cells that expressed a C-terminally truncated IL-13Ralpha2 chain (Delta335) did not bind IL-13, suggesting that the trileucine region modulates IL-13 binding. Truncation of IL-13Ralpha2 chain with a mutation in the trileucine region resulted in significantly decreased internalization compared with wild type IL-13Ralpha2 chain transfected cells. COS-7 cells transfected with tyrosine motif mutants exhibited a similar internalization level compared with wild type IL-13Ralpha2 chain transfected cells; however, dissociation of cell surface IL-13 was faster compared with wild type IL-13Ralpha2 transfectants. These results were further confirmed by determining the cytotoxicity of a chimeric protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR) to cells that expressed IL-13Ralpha2 chain mutants. We further demonstrate that the IL-13Ralpha2 chain is not ubiquitinated and that internalization of IL-13Ralpha2 did not depend on ubiquitination. Together, our findings suggest that the dileucine motif in the trileucine region and tyrosine motif participate in IL-13Ralpha2 internalization in distinct manners.     

The structure of the interleukin-15 alpha receptor and its implications for ligand binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.     

Crystal Structure of the interleukin-15.interleukin-15 receptor alpha complex: insights into trans and cis presentation

Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptor alpha (IL-15Ralpha) from one subset of cells presents IL-15 to neighboring IL-2Rbeta/gammac-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Ralpha. The structure reveals that the alpha receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Ralpha that account for the exceptionally high affinity of the IL-15.IL-15Ralpha complex. Interestingly, analysis of the topology of IL-15 and IL-15Ralpha at the IL-15.IL-15Ralpha interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Ralpha and IL-2Rbeta/gammac expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Ralpha permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15.IL-15Ralpha.IL-2Rbeta.gammac quaternary complex assembly through the use of molecular modeling.     

Expression of human IL-13 receptor alpha2 extracellular domain in Pichia pastoris

Interleukin-13 receptor alpha2 (IL-13Ralpha2) binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. We expressed the extracellular domain of human IL-13Ralpha2 (1-313) in methylotrophic yeast Pichia pastoris. SDS-PAGE analysis by PAS staining and Western blot analysis detected the product of the extracellular domain of human IL-13Ralpha2 as glycoprotein from P. pastoris. The yield of purified extracellular domain of human IL-13Ralpha2 was 2mg from 1L of culture. From CD analysis, the 2D structure of the purified IL-13Ralpha2 showed the typical beta-sheet. ELISA of the purified IL-13Ralpha2 detected the binding activity for human IL-13. Thus, it was found that the active extracellular domain of human IL-13Ralpha2 was expressed from P. pastoris.     

On-column refolding and characterization of soluble human interleukin-15 receptor alpha-chain produced in Escherichia coli

Interleukin-15 receptor alpha-chain (IL-15Ralpha) is a member of the new cytokine receptor family, which possesses the sushi domain. To investigate the biochemical and biophysical characteristics of soluble human IL-15Ralpha (shIL-15Ralpha), shIL-15Ralpha was recombinantly expressed in Escherichia coli. The shIL-15Ralpha containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni-nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration. The refolded shIL-15Ralpha exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Ralpha on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively. Thus, we succeeded in refolding shIL-15Ralpha to an active form on an affinity column.     

Internalization and nuclear localization of interleukin 1 are not sufficient for function.

The human fibroblast interleukin 1 (IL-1) receptor is a glycosylated transmembrane protein with a cytoplasmic domain of 213 amino acids. We have constructed a series of deletion mutants of the cytoplasmic region of the IL 1 receptor and have used these mutants to examine its role in ligand binding, internalization, signal transduction, and nuclear localization of IL-1. Mutant receptors lacking most of the cytoplasmic domain are expressed at the cell surface and can bind, internalize, and localize IL-1 at the nucleus, but they do not allow IL-1-mediated induction of interleukin 2 and SV40 promoters. We have localized a critical region for signal transduction to a 50-amino acid segment of the cytoplasmic domain of the receptor. These studies demonstrate that IL-1 internalization and nuclear localization are not sufficient to trigger IL-1 activation of gene expression in T-cells.     

Elucidation of the interleukin-15 binding site on its alpha receptor by NMR

The cytokine interleukin-15 (IL-15) signals through the formation of a quaternary receptor complex composed of an IL-15-specific alpha receptor, together with beta and gammac receptors that are shared with interleukin-2 (IL-2). The initiating step in the formation of this signaling complex is the interaction between IL-15 and IL-15Ralpha, which is a single sushi domain bearing strong structural homology to one of the two sushi domains of IL-2Ralpha. The crystal structure of the IL2-Ralpha/IL-2 complex has been determined, however little is known about the analogous IL-15Ralpha/IL-15 binding interaction. Here we show that recombinant IL-15 can be overexpressed as a stable complex in the presence of its high affinity receptor, IL-15Ralpha. We find that this complex is 10-fold more active than IL-15 alone in stimulating proliferation and survival of memory phenotype CD8 T cells. To probe the ligand/receptor interface, we used solution NMR to map chemical shifts on 15N-labeled IL-15Ralpha in complex with unlabeled IL-15. Our results predict that the binding surface on IL-15Ralpha involves strands C and D, similar to IL-2Ralpha. The interface, as predicted here, leaves open the possibility of trans-presentation of IL-15 by IL-15Ralpha on an opposing cell.     

Complex role of the IL-4 receptor alpha in a murine model of airway inflammation: expression of the IL-4 receptor alpha on nonlymphoid cells of bone marrow origin contributes to severity of inflammation

Recent studies have suggested the IL-4Ralpha expressed on lung epithelium is necessary for TH2-mediated goblet cell differentiation and mucus hypersecretion in a murine model of allergic lung disease. However, the IL-4Ralpha is expressed on numerous cell types that could contribute to the overall pathology and severity of asthma. The relative role of the receptor on these cells has not yet been conclusively delineated. To dissect the contribution of IL-4Ralpha in the development of pulmonary allergic responses, we generated murine radiation bone marrow (BM) chimeras. BM from IL-4Ralpha(+) or IL-4Ralpha(-) mice was transferred into recipient mice that expressed or lacked IL-4Ralpha. In the absence of IL-4Ralpha in recipient mice, there was no goblet cell metaplasia or mucus hypersecretion in response to OVA, even in the presence of TH2 cells and substantial eosinophilic infiltration. More importantly, we found that expression of the IL-4Ralpha on a nonlymphoid, MHC class II(+), BM-derived cell type contributes to the severity of inflammation and mucus production. These results suggest that IL-4 and IL-13 contribute to the development of allergic inflammation by stimulating a complex interaction between IL-4Ralpha(+) cell types of both bone marrow and non-bone marrow origin.     

IL-2 and IL-15 manifest opposing effects on activation of nuclear factor of activated T cells

IL-2 and IL-15 are cytokines involved in T cell activation and death. Their non-shared receptors, IL-2Ralpha and IL-15Ralpha, are important in the homeostasis of lymphocytes as evidenced by gene deletion studies. How these cytokine/receptor systems affect T cell antigen receptor signaling pathways is poorly understood. Here, we show that the IL-2 and IL-15 cytokine/receptor alpha systems regulate activation of nuclear factor of activated T cells (NF-AT) in opposing ways. IL-15Ralpha increased while IL-2Ralpha decreased basal NF-AT activation status in a Jurkat transient transfection model. The effect of each of the alpha chain receptors on NF-AT activation was further opposed by addition of the respective cytokine. These effects were inhibited by anti-cytokine and anti-cytokine receptor reagents as well as by inhibitors of TCR signaling. These results suggest a novel pathway of cytokine action to regulate T cell signaling, activation, death, and homeostasis.     

The IL-15R alpha chain signals through association with Syk in human B cells.

The alpha-chain of the IL-15R (IL-15Ralpha) serves as the specific, high-affinity receptor for IL-15. It is expressed by lymphoid and nonlymphoid cells, including B cell lymphoma lines. In this study, we have further explored IL-15Ralpha-mediated signaling in activated primary B cells and in Raji cells, a human B-lymphoblastoid cell line which expresses the IL-15Ralpha and IL-2Rgamma chains, but lacks the IL-2Rbeta chain. Stimulation of Raji cells with IL-15 induces their proliferation and rescues them from C2-ceramide-induced apoptosis. By immunoprecipitation and Western blotting, we show that treatment of Raji cells and activated primary B cells with IL-15 induces coprecipitation of Syk kinase with the IL-15Ralpha chain. Upon association, the activated Syk kinase phosphorylates the IL-15Ralpha chain as well as phospholipase Cgamma, which coprecipitates with Syk. Furthermore, transfection of Raji cells with stem-loop Syk antisense oligonucleotides prevents IL-15Ralpha and phospholipase Cgamma phosphorylation as well as the inhibition of apoptosis by IL-15. Mutation of a defined region of the intracellular signaling portion of IL-15Ralpha (Tyr227) abrogates both the IL-15Ralpha/Syk association and IL-15Ralpha phosphorylation. Taken together, this suggests that Syk kinase physically and functionally associates with the IL-15Ralpha chain in B cells and that Syk plays a key role in mediating IL-15-induced signal transduction, thus accounting for the distinct functional consequences of IL-15 vs IL-2 binding to B cells.     

Death deflected: IL-15 inhibits TNF-alpha-mediated apoptosis in fibroblasts by TRAF2 recruitment to the IL-15Ralpha chain.

Interleukin-15 (IL-15) is a potent inhibitor of several apoptosis pathways. One prominent path toward apoptosis is the ligand-induced association of TNF receptor 1 (TNFR1) with death domain adaptor proteins. Studying if and how IL-15 blocks TNFR1-mediated apoptosis in a murine fibroblast cell line (L929), we show here that IL-15 blocks TNFR1-induced apoptosis via IL-15Ralpha chain signaling. The intracellular tail of IL-15Ralpha shows sequence homologies to the TRAF2 binding motifs of CD30 and CD40. Most important, binding of IL-15 to IL-15Ralpha successfully competes with the TNFR1 complex for TRAF2 binding, which may impede assembly of key adaptor proteins to the TNFR1 complex, and induces IkappaBalpha phosphorylation. Thus, IL-15Ralpha chain stimulation is a powerful deflector of cell death very early in the apoptosis signaling cascade, while TNF-alpha and IL-15 surface as major opponents in apoptosis control.     

Soluble Interleukin IL-15Ralpha is generated by alternative splicing or proteolytic cleavage and forms functional complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.