The Structure of the Glial Cell Line-derived Neurotrophic Factor-Coreceptor Complex: INSIGHTS INTO RET SIGNALING AND HEPARIN BINDING

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor α1 (GFRα1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF2·GFRα12 complex structure at 2.35 Å resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF2·GFRα12 complex and the previously published artemin2·GFRα32 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFRα interface between the two structures, in particular ones that buttress the key conserved ArgGFRα-Gluligand-ArgGFRα interaction. Second, the flexible GDNF ligand “finger” loops fit differently into the GFRαs, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand2-co-receptor2-RET2 heterohexamer complexes. Consistent with this, we showed that GDNF2·GFRα12 and artemin2·GFRα32 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFRα1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg190-Lys202 region in GFRα1 domain 2. This may explain how GDNF·GFRα1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.