Identification of the Key Amino Acids of Glial Cell Line-derived Neurotrophic Factor Family Receptor α1 Involved in Its Biological Function

Abstract

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor α1 (GFRα1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRal function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRα1 mutations was made, and PC12 cell lines stably expressing different GFRα1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRaα1-Ret interaction assay, residues 152NN 153, Arg259, and 316SNS318 in the GFRα1 central region were found to be critical for GFRα1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRα1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRα1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRα1 may be involved in binding to GDNF and Ret.