Syndecan-3 may act alone or as a coreceptor with RET to promote cell spreading, neurite outgrowth, and migration of cortical neurons by GNDF, NRTN, and ARTN.

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...

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.