Direct interactions among Ret, GDNF and GFRα1 molecules reveal new insights into the assembly of a functional three-protein complex

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 alpha 1 (GFR alpha 1) 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 GFR alpha 1 molecules. This procedure allowed us to identify distinct regions of Ret that are physically engaged in the interaction with GDNF and GFR alpha 1. The lack of these regions in a recombinant Ret form results in the failure of both structural and functional binding of Ret to GFR alpha 1/GDNF complex. Furthermore, a model for the assembly of a transducing-competent Ret complex is suggested. (c) 2004 Elsevier Inc. All rights reserved.