Direct interactions among the tyrosine kinase receptor Ret, the GDNF and the GFR±1 molecules.

The glial-cell-line-derived neurotrophic factor (GDNF) ligand activates the Ret receptor through the assembly of a multiprotein complex, including the GDNF family receptor a1 (GFRa1) molecule. Given the neuroprotective role of GDNF, there is an obvious need to precisely identify the structural regions engaged in direct interactions between the three molecules. Here, we combined a functional approach for Ret activity (in PC12 cells) to cross-linking experiments followed by MS-MALDI to study the interactions among the purified extracellular region of the human Ret, GDNF and GFRa1 molecules. This procedure allowed us to identify distinct regions of Ret that are physically engaged in the interaction with GDNF and GFRa1. The lack of these regions in a recombinant Ret form results in the failure of both structural and functional binding of Ret to GFRa1/GDNF complex. Furthermore, a model for the assembly of a transducing-competent Ret complex is suggested.