The sensitivity of activated Cys Ret mutants to glial cell line-derived neurotrophic factor is mandatory to rescue neuroectodermic cells from apoptosis

Hirschsprung's disease (HSCR), a frequent developmental defect of the enter ic nervous system is due to loss-of-function mutations of RET, a receptor t yrosine kinase essential for the mediation of glial cell-derived neurotroph ic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutat ions (e.g., Cys(609), Cys(620), and Cys(634)) of the same gene are responsi ble for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by c ausing a covalent Ret dimerization, leading to ligand-independent activatio n of its tyrosine kinase. In this context, the association of Cys(609)- or Cys(620)-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys(634) Re t variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here t hat despite their constitutively activated kinase, the mere expression of t hese three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys(634) Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys(609) or Cys(620) mutat ions, which impair the terminal Ret glycosylation required for its insertio n at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together , these data support the idea that sensitivity to GDNF is the mandatory con dition, even for constitutively activated Ret mutants, to rescue neuroectod ermic cells from apoptosis. These findings may help clarify how a gain-of-f unction mutation can be associated with a developmental defect.