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MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) are dominantly inherited cancer syndromes. MEN2A is defined by the presence of medullary thyroid carcinoma, pheochromocytoma, and hyperparathyroidism. In addition to these abnormalities, MEN2B is characterized by skeletal abnormalities, ganglioneuromas of the intestinal tract, and mucosal neuromas. All three syndromes are associated with germline mutations of the RET proto-oncogene (1, 2), which codes for a receptor-like tyrosine kinase (3) whose ligand has not yet been identified. In the case of MEN2A and FMTC, point mutations result in the substitution of one of five Cys residues in the extracellular domain of RET (1). In MEN2B, a T(character omitted)C transition in the tyrosine kinase domain causes a Met(character omitted)Thr substitution at position 918 (2). It is not known how these alterations contribute to neoplasia. In the simplest model, the mutated RET allele acts as a dominant transforming gene. Alternatively, the mutations may inactivate a possible tumor suppressor function of proto-RET. In this scenario, the reduced dosage of proto-RET might be sufficient to trigger transformation or the product of the mutated allele might inhibit the function of the wild-type protein, thus exerting a dominant negative function (4.
To test these possibilities, we engineered eukaryotic expression vectors (5) encoding proto-RET (LTR-ret), three different MEN2A alleles (RET Cys sup 634 (character omitted)Tyr, Arg, or Trp; MEN2AY, MEN2AR, and MEN2AW, respectively), and the MEN2B allele (RET Met sup 918 (character omitted)Thr; MEN2B). After transfection in NIH 3T3 fibroblasts, RET mutants, but not proto-RET, displayed high transforming efficiency (Fig. 1).(Figure 1 omitted) In addition, MEN2A and MEN2B transfectants displayed high clonogenic ability in soft agar, whereas proto-RET transfectants did not show growth (6).
Mass populations of transfectants were obtained by killer HAT selection and used for protein analysis. RET was detected in two forms, one == 145 kD and the other == 160 kD (Fig. 2A), in all of the transfectants (7).(Figure 2A omitted) Comparable amounts of RET were then immunoprecipitated from the various transfectants and assayed for phosphotyrosine (pTyr) content (8), a hallmark of receptor autophosphorylation and activation. No tyrosine phosphorylation of the proto-RET product was detectable, whereas RET-MEN2A and RET-MEN2B products showed high amounts of pTyr (Fig. 2A). Similar results were obtained in immunocomplex kinase assays (9). The proto-RET product displayed little,...