Molecular dissection of human interleukin-31-mediated signal transduction through site-directed mutagenesis.

Interleukin (IL)-31 is a recently described cytokine, preferentially produced by T helper 2 lymphocytes and associated with skin diseases, such as atopic dermatitis. IL-31 is a member of the four alpha-helix bundle cytokine family and is related to the IL-6 subgroup. Its heterodimeric membrane receptor is composed of the gp130-like receptor (GPL) subunit associated to the oncostatin M receptor subunit. We identified critical amino acids implicated in the ligand receptor interaction by computational analysis combined with site-directed mutagenesis. Six IL-31 residues selected for their putative involvement in cytokine receptor contact sites were alanine-substituted, and the corresponding proteins were expressed in mammalian and bacterial systems. Biochemical, membrane binding, cell signaling, and cell proliferation analyses showed that mutation E44A, E106A, or H110A abolished IL-31 binding to GPL and the subsequent signaling events. A second ligand receptor-binding site involved Lys(134), with alanine substitution leading to a protein that still binds GPL, but is unable to recruit the second receptor subunit and the subsequent signaling pathways. The results indicate that IL-31 recognizes its receptor complex through two different binding sites, and we propose a three-dimensional model for IL-31.

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on the ciliary neurotrophic factor receptor alpha chain.

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin are members of the four-helix bundle cytokine family. These proteins signal through a common tripartite receptor composed of leukemia inhibitory factor receptor, gp130, and ciliary neurotrophic factor receptor alpha. Binding to ciliary neurotrophic factor receptor alpha occurs through an interaction site located at the C terminus of the cytokine AB loop and alphaD helix, known as site 1. In the present study, we have generated a model of neuropoietin and identified a conserved binding site for the three cytokines interacting with ciliary neurotrophic factor receptor alpha. To identify the counterpart of this site on ciliary neurotrophic factor receptor alpha, its cytokine binding domain was modeled, and the physicochemical properties of its surface were analyzed. This analysis revealed an area displaying properties complementary to the site 1 of ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin. Based on our computational predictions, residues were selected for their potential involvement in the ciliary neurotrophic factor receptor alpha binding epitope, and site-directed mutagenesis was carried out. Biochemical, cell proliferation, and cell signaling analyses showed that Phe(172) and Glu(286) of ciliary neurotrophic factor receptor alpha are key interaction residues. Our results demonstrated that ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on ciliary neurotrophic factor receptor alpha.

Neuropoietin, a new IL-6-related cytokine signaling through the ciliary neurotrophic factor receptor.

A structural profile-based computational screen was used to identify neuropoietin (NP), a new cytokine. The np gene is localized in tandem with the cardiotrophin-1 gene on mouse chromosome 7. NP shares structural and functional features with ciliary neurotrophic factor (CNTF), cardiotrophin-1, and cardiotrophin-like cytokine. It acts through a membrane receptor complex comprising CNTF receptor-alpha component (CNTFRalpha), gp130, and leukemia inhibitory factor receptor to activate signal transducer and activator of transcription 3 signaling pathway. NP is highly expressed in embryonic neuroepithelia. Strikingly, CNTFRalpha, but not its alternate ligands, CNTF and cardiotrophin-like cytokine, is expressed at the same developmental stages. NP is also observed in retina and to a lesser extent in skeletal muscle. Moreover, NP could sustain the in vitro survival of embryonic motor neurons and could increase the proliferation of neural precursors when associated to epidermal growth factor and fibroblast growth factor 2. Thus, NP is a new ligand for CNTFRalpha, with important implications for murine nervous system development.

Predominant expression of the long isoform of GP130-like (GPL) receptor is required for interleukin-31 signaling.

Gp130-like receptor (GPL) is a newly identified cytokine receptor. A recent study reported the involvement of GPL, together with OSMR, in the formation of the receptor complex for IL-31, a novel immune cytokine with a skin tropism. In the present work, we analyzed the signaling properties of IL-31 in glioblastoma and melanoma tumor cells. We demonstrate that in response to IL-31, its receptor complex recruits Jak1, Jak2, STAT1, -3, -5 signaling pathways, as well as the Pi3 kinase / AKT cascade. SHP-2 and Shc adapter molecules are also recruited and contribute to an increased activation of the MAP kinase pathway in response to IL-31. Different responses were observed depending on the expression of short or long GPL receptor isoform within the studied cell lines. We show that the short form of GPL receptor exerts a profound inhibitory effect on the signaling of IL-31 and behaves as a dominant negative receptor.