GRB2 BINDING TO THE DIFFERENT ISOFORMS OF RET TYROSINE KINASE

The RET proto-oncogene encodes two isoforms of a receptor tyrosine kin ase which plays a role in neural crest and kidney development. Ret lig ands have been recently identified as the neuron survival factor GDNF (Glial-Derived Neurotrophic Factor) and Neurturin. Somatic rearrangeme nts of RET, designated RET/PTCs, have been frequently detected in papi llary 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 Thy roid Carcinomas) as well as with the congenital megacolon or Hirschspr ung's disease, thus enlightening a significant role of this receptor g ene in diverse human pathologic conditions. In this study, by performi ng classical inhibition experiments using synthetic phosphopeptides an d by site-directed mutagenesis of the putative docking site, we have d etermined that for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform (corresponding to Tyr 1096 on proto-Ret). Howeve r, in intact cells, the interaction of Grb2 with the two short and lon g Ret isoforms expressed separately is of similar strength, thus sugge sting that Ret short isoform interaction with Grb2 could be mediated n ot only by Shc but also by a molecule that binds preferentially to thi s isoform. This possibility is supported by the evidence that the muta nt Ret/ptc2Y620F long isoform displays a weak coimmunoprecipitation wi th Grb2 and that this mutant, lacking the docking site for Grb2 but ow ing all the others phosphotyrosines, surprisingly displays a reduced t ransforming activity compared to that of the two WTs oncogenes. We thu s conclude that in intact cells both Ret isoforms bind to Grb2, althou gh with different modalities, In addition, the present results are in agreement with the possibility that different signal transduction path ways are associated with the two isoforms of Ret.