ALANINE SUBSTITUTION FOR THR(268) AND ASP(269) OF SOLUBLE CILIARY NEUROTROPHIC FACTOR (CNTF) RECEPTOR-ALPHA COMPONENT DEFINES A SPECIFIC ANTAGONIST FOR THE CNTF RESPONSE

Ciliary neurotrophic factor (CNTF) associates with an cu subunit (CNTF R alpha) of the receptor complex to initiate signal transduction by fa cilitating heterodimerization of the gp130 transducing protein and the leukemia inhibitory factor receptor (LIFR) beta. CNTFR alpha is ancho red to the membrane by a glycosylphosphatidylinositol Linkage; however , a soluble form of the alpha subunit can still bind CNTF to recruit t he signal transducing components of the receptor complex. In the prese nt study me show that alanine substitution for residues Thr(268) and A sp(269) of the CNTFR alpha subunit results in a mutated receptor subun it (R3), which can bind CNTF with an affinity similar to that of the w ild type CNTFR alpha but, when expressed as a soluble receptor subunit , lowers the binding of CNTF to its tripartite receptor. In addition, CNTFR3 alpha inhibits the proliferation of the TF1 hematopoietic cell line triggered by CNTF plus soluble wild type CNTFR alpha but not by I L-6 or oncostatin M. Similarly, CNTFR3 alpha specifically antagonizes the induction of gp130 and LIFR beta tyrosine phosphorylation observed in response to CNTF and wild type soluble CNTFR alpha in the HepG2 he patoma cell line, as well as the subsequent events leading to haptoglo bin synthesis. Positions 268 and 269 of CNTFR alpha appear to be criti cal for its interaction with gp130 and LIFR beta, whereby alanine subs titution of the residues at these positions results in antagonism of t he CNTF-induced response.