title: Identification of the key amino acids of glial cell line-derived neurotrophic factor family receptor alpha1 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 alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 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 GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.
authors.affiliation: Department of Neurobiology, the Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China.
authors.forename: Li-Mei
authors.initials: LM
authors.lastname: Wang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhu
authors.forename: Cheng
authors.initials: C
authors.lastname: He
authors.forename: Chang-Lin
authors.initials: CL
authors.lastname: Lu
authors.forename: Da-Fu
authors.initials: DF
authors.lastname: Ding
authors.forename: Zhe-Yu
authors.initials: ZY
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
chemical_list: C495041:Gdnf protein, rat; C494989:Gfra1 protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C495001:Ret protein, rat