Alanine Substitution for Thr268 and Asp269 of Soluble Ciliary Neurotrophic Factor (CNTF) Receptor α Component Defines a Specific Antagonist for the CNTF Response

Abstract Ciliary neurotrophic factor (CNTF) associates with an α subunit (CNTFRα) of the receptor complex to initiate signal transduction by facilitating heterodimerization of the gp130 transducing protein and the leukemia inhibitory factor receptor (LIFR) β. CNTFRα is anchored to the membrane by a glycosylphosphatidylinositol linkage; however, a soluble form of the α subunit can still bind CNTF to recruit the signal transducing components of the receptor complex. In the present study we show that alanine substitution for residues Thr268 and Asp269 of the CNTFRα subunit results in a mutated receptor subunit (R3), which can bind CNTF with an affinity similar to that of the wild type CNTFRα but, when expressed as a soluble receptor subunit, lowers the binding of CNTF to its tripartite receptor. In addition, CNTFR3α inhibits the proliferation of the TF1 hematopoietic cell line triggered by CNTF plus soluble wild type CNTFRα but not by IL-6 or oncostatin M. Similarly, CNTFR3α specifically antagonizes the induction of gp130 and LIFRβ tyrosine phosphorylation observed in response to CNTF and wild type soluble CNTFRα in the HepG2 hepatoma cell line, as well as the subsequent events leading to haptoglobin synthesis. Positions 268 and 269 of CNTFRα appear to be critical for its interaction with gp130 and LIFRβ, whereby alanine substitution of the residues at these positions results in antagonism of the CNTF-induced response.