Identification of a surface for binding to the GDNF-GFRα1 complex in the first cadherin-like domain of RET

The RET receptor tyrosine kinase is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GDNF-family receptor-alpha (GFRalpha) glycosylphosphatidylinositol-linked co-receptor. Molecular modeling studies of the extracellular domain of RET (RETECD) have revealed the existence of four cadherin-like domains (CLD1-4) followed by a cysteine-rich domain. Cross-linking experiments have indicated that the RETECD makes direct contacts with both the GDNF ligand and GFRalpha1 molecule in the complex, although it has low or no detectable affinity for either component alone. We have exploited sequence and functional divergences between the ectodomains of mammalian and amphibian RET molecules to map binding determinants in the human RETECD responsible for its interaction with the GDNF-GFRalpha1 complex by homologue-scanning mutagenesis. We found that Xenopus RETECD was unable to bind to GDNF-GFRalpha-1 or neurturin (NTN)-GFRalpha-2 complexes of mammalian origin. However, a chimeric molecule containing CLD1, - 2, and - 3 from human RETECD, but neither domain alone, had similar binding activity as compared with wild type human RETECD, suggesting the existence of an extended ligand binding surface within the three N-terminal cadherin-like domains of human RETECD. Subsequent loss-of-function experiments at higher resolution identified three small subsets of residues, mapping on the same face of the molecular model of RET CLD1, that were required for the interaction of human RETECD with the GDNF-GFRalpha1 complex. Additional experiments demonstrated that N-linked glycosylation of human RETECD was not required for ligand binding. Based on these observations, we propose a model for the assembly and architecture of the GDNF-GFRalpha1-RET complex.