Binding Characteristics of Ciliary Neurotrophic Factor to Sympathetic Neurons and Neuronal Cell Lines

CNTF and LIF treatments down-regulated CNTF binding to sympathetic neurons and EW-1 cells, suggesting that heterologous ligands can regulate CNTF receptor levels, which may in turn modulate the efficacy of CNTF in vitro and in vivo.

Abstract

Ciliary neurotrophic factor (CNTF) is a cytokine whose actions are largely restricted to the nervous system because of the predominant neuronal distribution of its receptor, CNTFRα. In this study, we sought to define the binding characteristics of CNTF to cultured sympathetic neurons and cell lines of neuronal origin. We report that 125I-CNTF binds to cultured sympathetic neurons, MAH, PC12, and EW-1 cells via high and low affinity receptors that can be distinguished on the basis of their dissociation constants (K D1∼10−12M and K D2∼10−9M). Competition experiments showed that the IC 50 for rat and human CNTF were, respectively, 65 pM and 5 nM for sympathetic neurons and 75 pM and 1.2 nM for EW-1 cells. Interestingly, leukemia inhibitory factor (LIF) did not compete for CNTF binding even at 100 nM concentration. The binding of 125I-CNTF to sympathetic neurons involved all three components of the CNTF receptor complex, namely CNTFRα, LIFR, and gp130, as shown by cross-linking experiments. CNTF and LIF treatments down-regulated CNTF binding to sympathetic neurons and EW-1 cells, suggesting that heterologous ligands can regulate CNTF receptor levels, which may in turn modulate the efficacy of CNTF in vitro and in vivo. Ciliary neurotrophic factor (CNTF) is a cytokine whose actions are largely restricted to the nervous system because of the predominant neuronal distribution of its receptor, CNTFRα. In this study, we sought to define the binding characteristics of CNTF to cultured sympathetic neurons and cell lines of neuronal origin. We report that 125I-CNTF binds to cultured sympathetic neurons, MAH, PC12, and EW-1 cells via high and low affinity receptors that can be distinguished on the basis of their dissociation constants (K D1∼10−12M and K D2∼10−9M). Competition experiments showed that the IC 50 for rat and human CNTF were, respectively, 65 pM and 5 nM for sympathetic neurons and 75 pM and 1.2 nM for EW-1 cells. Interestingly, leukemia inhibitory factor (LIF) did not compete for CNTF binding even at 100 nM concentration. The binding of 125I-CNTF to sympathetic neurons involved all three components of the CNTF receptor complex, namely CNTFRα, LIFR, and gp130, as shown by cross-linking experiments. CNTF and LIF treatments down-regulated CNTF binding to sympathetic neurons and EW-1 cells, suggesting that heterologous ligands can regulate CNTF receptor levels, which may in turn modulate the efficacy of CNTF in vitro and in vivo.