Background & Aims: Many missense mutations in the RET proto-oncogene were found in familial and sporadic cases of Hirschsprung disease (HSCR). The aim of this study was to make clear the mechanisms of RET dysfunction by HSCR mutations identified in its kinase domain. Methods: Ten kinase domain HSCR mutations were introduced into wild-type RET and constitutively activated RET with a multiple endocrine neoplasia 2A mutation, and the resulting mutant complementary DNAs were transfected into SK-N-MC primitive neuroectodermal tumor cells or NIH 3T3 fibroblast cells. The levels of activation of mutant RET and representative signaling molecules were investigated in the transfectants. Results: E762Q, S767R, R972G, and M980T mutations partially impaired the RET kinase activity and the representative signaling pathways. In particular, these mutations severely impaired the phospholipase C-γ signaling pathway in SK-N-MC cells. S765P, R873Q, F893L, R897Q, and E921K mutations resulted in a complete loss of the RET kinase activity. The P973L mutation markedly decreased the expression of the RET protein with normal kinase activity. Conclusions: Hirschsprung disease can result from these distant functional classes of kinase domain mutation of RET.
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