Functional mapping of receptor specificity domains of glial cell line-derived neurotrophic factor (GDNF) family ligands and production of GFRα1 RET-specific agonists

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor alpha-component (GFR alpha 1-GFR alpha 4)) and the receptor tyrosine kinase RET, To bean characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFR alpha 1.RET or GFR alpha 2.RET) identified sites along the second finger of GDNF critical for activating the GFR alpha 1.RET and GFR alpha 2.RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFR alpha 1.RET, but additional determinants are required for interaction with the other GFR alpha s, This difference in the molecular basis of GFL-GFR alpha specificity allowed the production of GFR alpha 1.RET-specific agonists and provides a foundation for understanding of GFL-GFR alpha.RET signaling at the molecular level.