Direct interactions among Ret, GDNF and GFRalpha1 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 alpha1 (GFRalpha1) 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 GFRalpha1 molecules. This procedure allowed us to identify distinct regions of Ret that are physically engaged in the interaction with GDNF and GFRalpha1. The lack of these regions in a recombinant Ret form results in the failure of both structural and functional binding of Ret to GFRalpha1/GDNF complex. Furthermore, a model for the assembly of a transducing-competent Ret complex is suggested.
Amoresano
Angela
A
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Montesantangelo, via Cinthia 4, 80126 Naples, Italy.
Incoronato
Mariarosaria
M
Monti
Gianluca
G
Pucci
Piero
P
de Franciscis
Vittorio
V
Cerchia
Laura
L
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009414:Nerve Growth Factors / Q000737:chemistry / Q000378:metabolism*; D015513:Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C495041:Gdnf protein, rat; C494989:Gfra1 protein, rat; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D009414:Nerve Growth Factors; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D020794:Receptor Protein-Tyrosine Kinases; C495001:Ret protein, rat