VARIOUS MECHANISMS CAUSE RET-MEDIATED SIGNALING DEFECTS IN HIRSCHSPRUNGS-DISEASE

Hirschsprung's disease (HSCR) is a common congenital malformation char acterized by the absence of intramural ganglion cells of the hindgut. Recently, mutations of the RET tyrosine kinase receptor have been iden tified in 50 and 15-20% of familial and sporadic HSCR, respectively. T hese mutations include deletion, insertion, frameshift, nonsense, and missense mutations dispersed throughout the RET coding Sequence. To in vestigate their effects on RET function, seven HSCR missense mutations were introduced into either a 1114-amino acid wild-type RET isoform ( RET51) or a constitutively activated form of RET51 (RET-MEN 2A). Here, we report that one mutation affecting the extracytoplasmic cadherin d omain (R231H) and two mutations located in the tyrosine kinase domain (K907E, E921K) impaired the biological activity of RET-MEN 2A when tes ted in Rat1 fibroblasts and pheochromocytoma PC12 cells. However, the mechanisms resulting in RET inactivation differed since the receptor b earing R231H extracellular mutation resulted in an absent RET protein at the cell surface while the E921K mutation located within the cataly tic domain abolished its enzymatic activity, In contrast, three mutati ons mapping into the intracytoplasmic domain neither modified the tran sforming capacity of RET-MEN 2A nor stimulated the catalytic activity of RET in our ligand-independent system (S767R, P1039L, M1064T). Final ly, the C609W HSCR mutation exerts a dual effect on RET since it leads to a decrease of the receptor at the cell surface and converted RET51 into a constitutively activated kinase due to the formation of disulf ide-linked homodimers. Taken together, our data show that allelic hete rogeneity at the RET locus in HSCR is associated with various molecula r mechanisms responsible for RET dysfunction.