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The ret oncogene frequently has been found activated in papillary thyroid carcinomas. A previous characterization of ret activation revealed recombination of its tyrosine kinase domain and sequences derived from an uncharacterized locus (DlOS170). The mechanism leading to this recombination was identified as a paracentric inversion of the long arm of chromosome 10, inv(10)(qll.2q21), with the breakpoints occurring where ret and DlOS170 were mapped. To further characterize the activation of ret in papillary thyroid carcinomas, we have now isolated and sequenced a second type of ret oncogenic rearrangement not involving the DlOS170 locus. The nucleotide sequence indicated that the transforming activity was created by the fusion of the ret tyrosine kinase domain with part of the Rle regulatory subunit of protein kinase A (PKA). This is the first example of an oncogenic activity involving a PKA gene. PKA is the main intracellular cyclic AMP receptor, and its RIax subunit gene is located on chromosome 17q. Rlo-ret transcripts encode two isoforms of the chimeric protein (p76 and p81), which display constitutive tyrosine phosphorylation as well as a tyrosine kinase enzymatic activity. Under nonreducing conditions, both isoforms are found in a dimeric configuration because of both homo-and heterodimer formation. Thus, the in vivo activation of ret in human papillary thyroid carcinomas is provided by the fusion of its tyrosine kinase domain with different genes and can be mediated by different mechanisms of gene rearrangement.

We have introduced three Hirschsprung (HSCR) mutations localized in the tyrosine kinase domain of RET into the RET/PTC2 chimaeric oncogene which is capable of transforming NIH3T3 mouse fibroblasts and of differentiating pC12 rat pheochromocytoma cells. The three HSCR mutations abolished the biological activity of RET/PTC2 in both cell types and significantly decreased its tyrosine phosphorylation. By contrast, a rare polymorphism in exon 18 does not alter the transforming capability of RET/PTC2 or its tyrosine phosphorylation. These data suggest a loss of function effect of HSCR mutations which might act through a dominant negative mechanism. Our model system is therefore capable of discriminating between causative HSCR mutations and rare polymorphisms in the tyrosine kinase domain of RET.