Evidence for a ligand-specific signaling through GFR alpha-1, but not GFR alpha-2, in the absence of ret

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two homologeous proteins that have been recognized as potent survival facto rs for distinct neuronal populations. GDNF and NTN act through a two-compon ent receptor system consisting of the ligand-specific binding subunits GDNF family receptor (GFR)alpha -1 and GFR alpha -2 and the common transducing subunit c-Ret. In addition, it has been demonstrated that GDNF can signal t hrough GFR alpha -1 in the absence of c-Ret. In the present study, we sough t to determine whether a similar c-Ret-independent signaling applies for GF R alpha -2. In addition, we have characterized the ligand specificity of th e c-Ret-independent action of GFR alphas. To establish an assay system for these studies, several neural cell lines were screened for the presence of GDNF and NTN receptor subunits by RT-PCR and immunoblot analysis. c-Ret exp ression was detectable only in Neuro2A cells, which did not express GFR alp ha -1 or GFR alpha -2. The neuronal cell line LS expressed GFR alpha -2, an d the glial cell line Mes42 expressed GFR alpha -1, whereas the neuronal ce ll line B104 expressed both GFR alpha -1 and GFR alpha -2. Stimulation of B 104 and Mes42 cells with GDNF, but not with NTN, for 10 min resulted in CRE B phosphorylation. In apparent contrast, neither NTN nor GDNF promoted CREB activation in LS and Neuro2A cells. Moreover, exposure of LS cells to NTN or GDNF also failed to activate AKT and ERK. Together these findings provid e evidence that, in contrast to GFR alpha -1, GFR alpha -2 fails to signal in the absence of c-Ret. In addition, these observations reveal that c-Ret- independent signaling of GFR alpha -1 is ligand-specific and occurs only wi th GDNF, (C) 2001 Wiley-Liss, Inc.