Identification Of Critical Residues Of GFRα1 Binding To GDNF

Glial cell line-derived neurotrophic factor (GDNF) was originally isolated based on its potent and specific ability to promote the survival and morphological differentiation of dopaminergic neurons and motoneurons in embryonic midbrain cultures. In addition, GDNF also support the survival and regulate the differentiation of many peripheral neurons, including sympathetic, parasympathetic, sensory and enteric neurons. GDNF also plays a crucial role outside the nervous system, as a morphogenetic factor in kidney development and as a regulator of spermatogonia differentiation. GDNF is an attractive therapeutic candidates for treatment of neurodegenerative diseases and nerve injuries. The neurotrophic and morphogenic activities of GDNF are mediated by its interaction with a multicomponent receptor complex formed by the RET receptor tyrosine kinase and a glycosylphosphatidylinositol (GPI)-anchored 'accessory' receptor, GDNF family receptor alpha-1 (GFRal). GFRal is an extracellular protein that is attached to the outer cell membrane by GPI-anchor and could not transducted signalling directly. According to the original model, a GDNF dimer first binds to either monomeric or dimeric GFRal, and the GDNF-GFRal complex then interacts with two RET molecules, thereby inducing their homodimerization and tyrosine autophosphorylation . However, the fact that GDNF mutants that are deficient in GFRal binding are able to activate RET indicates that at least some RET molecules are weakly associated with GFRal before GDNF binding and the existence of RET potentiates the affinity of GFRal binding to GDNF. The structure-function of GDNF and distinct structural elements in GDNF mediate binding to GFRal have been well documented. However, there are few works on structure and function analysis of GFRal, and the key residues of GFRal involved in interaction with GDNF have not been demonstrated yet. The knowledge of structural mechanism for the interaction between GFRal and GDNF will provide strong basis for designing small molecular peptide with same biological function of GDNF, which may be useful for treatment of neurodegenerative diseases and for further study the mechanism of otherneurotrophic factors.The PC12-cell line has been extensively used as a model for peripheral neuronal differentiation. In serum-containing medium, PC-12 cells undergo mitosis and display properties of adrenal chromaffin cells. Addition of NGF or FGF induces transformation of PC-12 cells to nondividing sympathetic neuron-like cells. Treatment with IGF- I or EGF instead stimulates proliferation of PC-12 cells but not differentiation. PC-12 cells does not express endogenous GFRal and only express low levels of RET.In this study, GFRal was amplified by RT-PCR and cloned into a pET vector. GFRal was high expressed in E.coli after IPTG induction. By Ni2+ chelation affinity chromatography, the purified recombinant GFRal protein were obtained. Based on the evolutionary trace method, multiple sequence alignment and dendrogram construction were then carried out by the ClustalX program and constructed a phylogenetic tree from a multiple sequence alignment for a protein family. Secondary structure and residues relative solvent accessibility predictions were carried out using PHD prediction program. The residues targeted for site-directed mutagenesis are in the central domain or C-terminal segments adjacent to the central domain of GFRal with relatively high solvent accessibility. These residues are mutated into alanine, either individually or in combinations of two to three residues. The mutants were transfected into PC 12 cells respectively to probed the ability respondence to GDNF stimulation and the interaction with RET by immunoprecipitation, c-Ret phosphorylation and immunoblotting assays.The main results are as follows: 1. Expression and purification of recombinant rat GFRal proteinTo obtain recombinant GFRal and study its biological activity, the cDNA encoding the mature rat GFRal was isolated using RT-PCR with total RNA extracted from newborn S...