G. Wang, L. Xiong, T. Gerassenkov, V. Kalabokis, A. Person
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) is one of three neurotrophic factor families found in the nervous system. The GFLs, along with their receptors, play important roles in axon guidance, synapse formation, neuronal survival, neural regeneration, and are implicated in a number of neurological diseases.1 There are four GFLs: GDNF, Neurturin, Artemin, and Persephin. These ligands are known to bind to the co-receptors GFRα-1, GFRα-2, GFRα-3, and GFRα-4, respectively, through which they activate the common signal transducer, the receptor tyrosine kinase RET (Rearranged during Transformation).2 A fifth coreceptor, GDNF Receptor-α-like (GFRAL) was identified in 2005, but the function of this orphan receptor is currently unknown.3 We have synthesized recombinant human GFRAL and tested the hypothesis that GFRAL interacts with known GFLs to change the dynamics of GFLs and their receptor networks. Both ELISA binding studies and cell-based assays were carried out using standard methods.4,5 Our results show that Neurturin binds to GFRAL with a high affinity (EC50 = 8 ng/mL, N=4). Artemin binds to GFRAL with a low affinity (EC50 = 2 mg/mL, N=2). GDNF and Persephin have only minimal or no binding to GFRAL (N=3). Addition of GFRAL proteins at a concentration of 0.5 µg/mL in cell culture media stimulated Neurturin induced cell proliferation in the SH-SY5Y human neuroblastoma cell line (EC50 of Neurturin = 0.5 µg/mL, N=4). This effect of GFRAL is similar to that of GFRα-2 (EC50 of Neurturin = 1 µg/mL, N=4), although the maximal response by GFRAL is lower than that of GFRα-2. GFRα-2 dose-dependently increased the effect of GFRAL in stimulating the Neurturin-induced cell proliferation in SH-SY5Y cells (N=2). On the contrary, addition of GFRAL did not affect the stimulatory effect of GFRα-2 on Neurturin-induced stimulation of SH-SY5Y cell proliferation (N=2). This is the first study to demonstrate that GFRAL interacts with GFLs, primarily Neurturin and Artemin, and can play a major role in GFLs and receptor networks involved in neuronal processes. These data also show that the biologically active recombinant GFRAL protein can be used to identify new GFRAL ligands and as a tool to investigate GDNF-related signaling mechanisms. The identification of this novel interaction between GFRAL and GDNF family ligands will enhance our understanding of the mechanisms and applications of GLFs and their receptors in neurodegenerative and neuropsychiatric diseases.