Abstract
Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor alpha1 (GFR alpha 1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF(2).GFR alpha 1(2) complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF(2).GFR alpha 1(2) complex and the previously published artemin(2).GFR alpha 3(2) complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR alpha interface between the two structures, in particular ones that buttress the key conserved Arg(GFR alpha)-Glu(ligand)-Arg(GFR alpha) interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR alphas, 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 ligand(2)-co-receptor(2)-RET(2) heterohexamer complexes. Consistent with this, we showed that GDNF(2).GFR alpha1(2) and artemin(2).GFR alpha 3(2) 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 GFR alpha 1 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 Arg(190)-Lys(202) region in GFR alpha 1 domain 2. This may explain how GDNF.GFR alpha 1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.
Highlights
Glial cell line-derived neurotrophic factor (GDNF),3 originally characterized as a growth factor promoting the survival of midbrain dopaminergic neurons [1], regulates the differentiation and development of many peripheral neurons [2] and is neuroprotective [3]
Biochemical and Structural Characterization of GDNF21⁄7 GFR␣12 Complex—We co-expressed GDNF family receptor ␣1 (GFR␣1) domain 2–3-C (GFR␣1 D23C) with GDNF in Tn5 insect cells and purified the GDNF21⁄7GFR␣12 complex in two steps: Ni2ϩ-Sepharose affinity purification in batch followed by size exclusion chromatography
SDS-PAGE showed that fraction 6 from the gel filtration column, which ran at about 100 kDa, contained two proteins, one of molecular mass 38 kDa (GFR␣1 D23C) and the other of molecular mass 22 kDa (GDNF) (Fig. 1A)
Summary
GDNF, originally characterized as a growth factor promoting the survival of midbrain dopaminergic neurons [1], regulates the differentiation and development of many peripheral neurons [2] and is neuroprotective [3]. Some clinical trials have indicated that perfusing GDNF into the putamen may be therapeutically beneficial in Parkinson disease [4] These neuroprotective and therapeutic roles have generated wide interest in the study of the GDNF signaling system. There are three other GDNF family ligands (GFLs), neurturin (NRTN [5]), artemin (ARTN [6]), and persephin (PSPN [7]), and knock-out mice experiments have made it clear that the order of biological importance is GDNF ϾϾ NRTN Ͼ ARTN Ͼ PSPN [2] They all signal primarily through the receptor tyrosine kinase RET [8]. GDNF in complex with GFR␣1 can signal through the neural cell adhesion molecule independent of RET [19], and recent studies suggest that the GDNF21⁄7GFR␣12 complex can act as an adhesin, mediating cell-cell interactions [20]. Our structure suggests how GDNF2-GFR␣12 interactions might lead to cell-cell adhesion and RET-independent signaling
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
