Amoresano Angela, Incoronato Mariarosaria, Monti Gianluca, Pucci Piero, de Franciscis Vittorio, Cerchia Laura
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Montesantangelo, via Cinthia 4, 80126 Naples, Italy.
Cell Signal. 2005 Jun;17(6):717-27. doi: 10.1016/j.cellsig.2004.10.012. Epub 2004 Nov 13.
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.
胶质细胞源性神经营养因子（GDNF）配体通过多蛋白复合物的组装激活Ret受体，该复合物包括GDNF家族受体α1（GFRα1）分子。鉴于GDNF的神经保护作用，显然需要精确鉴定参与这三种分子之间直接相互作用的结构区域。在此，我们将用于Ret活性的功能方法（在PC12细胞中）与交联实验相结合，随后进行MS-MALDI，以研究人Ret、GDNF和GFRα1分子纯化的细胞外区域之间的相互作用。该方法使我们能够鉴定出Ret中与GDNF和GFRα1相互作用的不同区域。重组Ret形式中这些区域的缺失导致Ret与GFRα1/GDNF复合物的结构和功能结合失败。此外，还提出了一种具有转导能力的Ret复合物组装模型。
