Ciliary neurotrophic factor (CNTF) is a member of the gp130 family of cytokines. The functional receptor complex of CNTF is composed of CNTF, the CNTF receptor α (CNTFR), gp130 and the leukemia inhibitory factor receptor (LIFR). Three regions on CNTF have been identified as binding sites for its receptors. The ligand-receptor interactions are mediated through the cytokine binding domains (CBD) and/or the immunoglobulin (Ig)-like domains of the receptors. However, in the case of CNTF, the precise nature of the protein-protein contacts in the signaling complex have not yet been resolved. The present study shows that the membrane distal CBD (CBD1), the membrane proximal CBD (CBD2) of LIFR, the CBD and Ig-like domain of gp130 associate in vitro with CNTF. LIFR CBD1 and gp130 CBD associate i...[ Read more ]

Ciliary neurotrophic factor (CNTF) is a member of the gp130 family of cytokines. The functional receptor complex of CNTF is composed of CNTF, the CNTF receptor α (CNTFR), gp130 and the leukemia inhibitory factor receptor (LIFR). Three regions on CNTF have been identified as binding sites for its receptors. The ligand-receptor interactions are mediated through the cytokine binding domains (CBD) and/or the immunoglobulin (Ig)-like domains of the receptors. However, in the case of CNTF, the precise nature of the protein-protein contacts in the signaling complex have not yet been resolved. The present study shows that the membrane distal CBD (CBD1), the membrane proximal CBD (CBD2) of LIFR, the CBD and Ig-like domain of gp130 associate in vitro with CNTF. LIFR CBD1 and gp130 CBD associate in vitro with soluble CNTFR in the absence of CNTF. Alanine substitution of Lys²¹⁹/ Leu²²⁰ in the B strand of the C-terminal (BC) module of the gp130 CBD and Leu¹⁵⁵/Lys¹⁵⁶ in the similar region of LIFR CBD1 reduced their binding to sCNTFR. Alanine substitiution of Thr²⁶⁸ and Asp²⁶⁹ in the E strand of BC module of CNTFR result in the loss of its binding capacity to gp130 CBD but not LIFR CBD1. Moreover, the CBD of gp130 competes with CBD1 of LIFR for the binding of soluble CNTFR and both domains partially block CNTF signaling, suggesting that they bind to the same site on CNTFR other than the E strand. More importantly, these soluble CBDs enhanced leukemia inhibitory factor (LIF) or IL-6 signaling in NT-2 cells. These data provide the first evidence that soluble CNTFR can independently bind to LIFR and gp130 in the absence of CNTF and raise the interesting possibility that CNTFR may pre-associate with the β receptor components of the CNTFR complex on the cell surface.

Having established the structural details of CNTFR complex, we try to study the putative cross-talks between gp130-LIFR signaling and neurotrophins in the process of neuronal differentiation. Nerve growth factor (NGF) is required for the development of sympathetic neurons and subsets of sensory neurons. Current knowledge on the molecular mechanisms underlying the biological function of NGF is mainly based on the studies with PC12 cells, which differentiate into sympathetic-like neurons with long neurite outgrowth upon NGF treatment. The present study demonstrates that expression of LIFR, but not gp130 and CNTFR, is gradually upregulated in PC12 cells when treated with NGF but not EGF. Attenuating LIFR signaling through stable transfection of antisense or dominant negative LIFR constructs enhanced NGF induced neurite extension in PC12 cells. On the contrary, overexpression of LIFR retarded the neurite outgrowth and induced cell clumping. These novel findings suggest that LIFR signaling, besides its known function for cell survival and neuronal cell type switch, can be specifically induced by NGF and exert negative regulatory effect on NGF-induced neurite outgrowth of PC12 cells.