Ciliary Neurotrophic Factor, Cardiotrophin-like Cytokine, and Neuropoietin Share a Conserved Binding Site on the Ciliary Neurotrophic Factor Receptor α Chain^*

Abstract

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 α. Binding to ciliary neurotrophic factor receptor α occurs through an interaction site located at the C terminus of the cytokine AB loop and αD 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 α. To identify the counterpart of this site on ciliary neurotrophic factor receptor α, 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 α binding epitope, and site-directed mutagenesis was carried out. Biochemical, cell proliferation, and cell signaling analyses showed that Phe¹⁷² and Glu²⁸⁶ of ciliary neurotrophic factor receptor α 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 α.