Grb2 binding to the different isoforms of Ret tyrosine kinase

The RET proto-oncogene encodes two isoforms of a receptor tyrosine kinase which plays a role in neural crest and kidney development. Ret ligands have been recently identified as the neuron survival factor GDNF (Glial-Derived Neurotrophic Factor) and Neurturin. Somatic rearrangements of RET, designated RET/PTCs, have been frequently detected in papillary thyroid carcinomas. In addition, distinct germ-line mutations of RET gene have been associated with the inherited cancer syndromes MEN (Multiple Endocrine Neoplasia) 2A, 2B and FMTC (Familial Medullar Thyroid Carcinomas) as well as with the congenital megacolon or Hirschsprung's disease, thus enlightening a significant role of this receptor gene in diverse human pathologic conditions. In this study, by performing classical inhibition experiments using synthetic phosphopeptides and by site-directed mutagenesis of the putative docking site, we have determined that for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform (corresponding to Tyr 1096 on proto-Ret). However, in intact cells, the interaction of Grb2 with the two short and long Ret isoforms expressed separately is of similar strength, thus suggesting that Ret short isoform interaction with Grb2 could be mediated not only by Shc but also by a molecule that binds preferentially to this isoform. This possibility is supported by the evidence that the mutant Ret/ptc2Y620F long isoform displays a weak coimmunoprecipitation with Grb2 and that this mutant, lacking the docking site for Grb2 but owing all the others phosphotyrosines, surprisingly displays a reduced transforming activity compared to that of the two WTs oncogenes. We thus conclude that in intact cells both Ret isoforms bind to Grb2, although with different modalities. In addition, the present results are in agreement with the possibility that different signal transduction pathways are associated with the two isoforms of Ret.