Determinants of ligand binding specificity in the glial cell line-derived neurotrophic factor family receptor αs

The glial cell line-derived neurotrophic factor (GDNF) family comprise a subclass of cystine-knot superfamily ligands that interact with a multisubunit receptor complex formed by the c-Ret tyrosine kinase and a cystine-rich glycosyl phosphatidylinositol-anchored binding subunit called GDNF family receptor alpha (GFR alpha). All four GDNF family ligands utilize c-Ret as a common signaling receptor, whereas specificity is conferred by differential binding to four distinct GFR alpha homologues. To understand how the different GFR alphas discriminate ligands, we have constructed a large set of chimeric and truncated receptors and analyzed their ligand binding and signaling capabilities. The major determinant of ligand binding was found in the most conserved region of the molecule, a central domain predicted to contain four conserved alpha helices and two beta strands. Distinct hydrophobic and positively charged residues in this central region were required for binding of GFR alpha1 to GDNF. Interaction of GFR alpha1 and GFR alpha2 with GDNF and neurturin required distinct subsegments within this central domain, which allowed the construction of chimeric receptors that responded equally well to both ligands, C-terminal segments adjacent to the central domain are necessary and have modulatory function in ligand binding. In contrast, the N-terminal domain was dispensable without compromising ligand binding specificity. Ligand-independent interaction with c-Ret also resides in the central domain of GFR alpha1, albeit within a distinct and smaller region than that required for ligand binding. Our results indicate that the central region of this class of receptors constitutes a novel binding domain for cystine-knot superfamily ligands.