Functional mapping of receptor specificity domains of glial cell line-derived neurotrophic factor (GDNF) family ligands and production of GFR alpha 1RET-specific agonists

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs ) (GDNF, neurturin, artemin, and persephin) are critical regulators of neur odevelopment and support the survival of midbrain dopaminergic and spinal m otor neurons in vitro and in animal disease models making them attractive t herapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affin ity binding component (GDNF-family receptor alpha-component (GFR alpha 1-GF R alpha 4)) and the receptor tyrosine kinase RET, To bean characterization of GFL receptor specificity at the molecular level, we performed comprehens ive homologue-scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from pe rsephin (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 activa ting the GFR alpha 1.RET and GFR alpha 2.RET receptor complexes. Furthermor e, 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 alp ha s, This difference in the molecular basis of GFL-GFR alpha specificity a llowed the production of GFR alpha 1.RET-specific agonists and provides a f oundation for understanding of GFL-GFR alpha.RET signaling at the molecular level.