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

The glial cell line-derived neurotrophic factor (GDNF) family comprise a su bclass of cystine-knot superfamily ligands that interact with a multisubuni t receptor complex formed by the c-Ret tyrosine kinase and a cystine-rich g lycosyl phosphatidylinositol-anchored binding subunit called GDNF family re ceptor alpha (GFR alpha). All four GDNF family ligands utilize c-Ret as a c ommon signaling receptor, whereas specificity is conferred by differential binding to four distinct GFR alpha homologues. To understand how the differ ent GFR alphas discriminate ligands, we have constructed a large set of chi meric and truncated receptors and analyzed their ligand binding and signali ng capabilities. The major determinant of ligand binding was found in the m ost conserved region of the molecule, a central domain predicted to contain four conserved alpha helices and two beta strands. Distinct hydrophobic an d positively charged residues in this central region were required for bind ing of GFR alpha1 to GDNF. Interaction of GFR alpha1 and GFR alpha2 with GD NF 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 a re necessary and have modulatory function in ligand binding. In contrast, t he N-terminal domain was dispensable without compromising ligand binding sp ecificity. Ligand-independent interaction with c-Ret also resides in the ce ntral domain of GFR alpha1, albeit within a distinct and smaller region tha n that required for ligand binding. Our results indicate that the central r egion of this class of receptors constitutes a novel binding domain for cys tine-knot superfamily ligands.