The Structure of the Glial Cell Line-derived Neurotrophic Factor-Coreceptor Complex

Abstract

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor ␣1 (GFR␣1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF 2 ⅐GFR␣1 2 complex structure at 2.35 Å resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF 2 ⅐GFR␣1 2 complex and the previously published artemin 2 ⅐GFR␣3 2 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFR␣ interface between the two structures, in particular ones that buttress the key conserved Arg GFR␣ -Glu ligand -Arg GFR␣ interaction. Second, the flexible GDNF ligand "finger" loops fit differently into the GFR␣s, 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 ⅐GFR␣1 2 and artemin 2 ⅐GFR␣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␣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␣1 domain 2. This may explain how GDNF⅐GFR␣1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET. 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). GDNF is also a morphogenic factor in kidney and spermatogonia development (reviewed by Airaksinen and Saarma (2)). 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 Ͼ Ͼ NRTN Ͼ ARTN Ͼ PSPN (2). They all signal primarily through the receptor tyrosine kinase RET (8). The extracellular region of RET has four cadherin-like domains and a cysteine-rich domain. Mutations in RET can cause both gain-of-function and loss-of-function diseases. In the former category are hereditary medullary thyroid carcinoma and multiple endocrine neoplasias of types 2A and 2B (9, 10), whereas Hirschsprung disease is an example of the latter (10).GFLs are distant relatives of transforming growth factor ␤ (2). Each GFL has its own co-receptor ␣: GDNF requires GFR␣1; N...

Structural basis of agrin–LRP4–MuSK signaling

“…First, agrin is a monomeric ligand, while the RET ligand GDNF functions as a homodimer (Wang et al 2006;Parkash et al 2008). Second, the formation of the agrin-LRP4 complex is a two-step synergistic process, while GFLs dimerize with GFRa in a 1:1 stoichiometry (Schlee et al 2006;Wang et al 2006;Parkash et al 2008). Finally, MuSK prebinds to its coreceptor, LRP4, before activation, while RET does not (Kim et al 2008;Zhang et al 2008).…”

“…However, key differences exist between the agrin-LRP4 and GFL-GFRa interactions. First, agrin is a monomeric ligand, while the RET ligand GDNF functions as a homodimer (Wang et al 2006;Parkash et al 2008). Second, the formation of the agrin-LRP4 complex is a two-step synergistic process, while GFLs dimerize with GFRa in a 1:1 stoichiometry (Schlee et al 2006;Wang et al 2006;Parkash et al 2008).…”

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

“…In addition, glial cell line-derived neurotrophic factor (GDNF), which is supplemented in our medium, was originally purified by heparin affinity chromatography (Lin et al, 1993) and has later been shown to interact with heparan sulfates (HSs; Rickard et al, 2003). HSs are required for GDNF signaling through the GFRα1-RET complex (Barnett et al, 2002;Parkash et al, 2008). The addition of heparin at a final concentration of 25-50 U/ml resulted in a more than twofold improvement in the yield of GFP-positive spermatogonia after 21 days of culture (Fig.…”

Assembly and activation of neurotrophic factor receptor complexes

“…Similar to other RTKs, Ret functions as a homodimer. In agreement with the two-fold symmetry of the ligands (Eigenbrot and Gerber, 1997), crosslinking and high-resolution structural studies have shown that GFRas also interact with GDNF proteins as homodimers (Jing et al, 1996;Trupp et al, 1998;Wang et al, 2006;Parkash et al, 2008). Association between Ret and GFRas has also been detected (Sanicola et al, 1997;Eketjäll et al, 1999;Cik et al, 2000), so the functional receptor complex for GDNF ligands is therefore likely to conform to a 2:2:2 stoichiometry [ Fig.…”