The Sensitivity of Activated Cys Ret Mutants to Glial Cell Line-Derived Neurotrophic Factor Is Mandatory To Rescue Neuroectodermic Cells from Apoptosis

Abstract

Hirschsprung's disease (HSCR), a frequent developmental defect of the enteric nervous system is due to loss-of-function mutations of RET, a receptor tyrosine kinase essential for the mediation of glial cell-derived neurotrophic factor (GDNF)-induced cell survival. Instead, gain-of-function Cys mutations (e.g., Cys⁶⁰⁹, Cys⁶²⁰, and Cys⁶³⁴) of the same gene are responsible for thyroid carcinoma (MEN2A/familial medullary thyroid carcinoma) by causing a covalent Ret dimerization, leading to ligand-independent activation of its tyrosine kinase. In this context, the association of Cys⁶⁰⁹- or Cys⁶²⁰-activating mutations with HSCR is still an unresolved paradox. To address this issue, we have compared these two mutants with the Cys⁶³⁴ Ret variant, which has never been associated with HSCR, for their ability to rescue neuroectodermic cells (SK-N-MC cells) from apoptosis. We show here that despite their constitutively activated kinase, the mere expression of these three mutants does not allow cell rescue. Instead, we demonstrate that like the wild-type Ret, the Cys⁶³⁴ Ret variant can trigger antiapoptotic pathways only in response to GDNF. In contrast, Cys⁶⁰⁹ or Cys⁶²⁰ mutations, which impair the terminal Ret glycosylation required for its insertion at the plasma membrane, abrogate GDNF-induced cell rescue. Taken together, these data support the idea that sensitivity to GDNF is the mandatory condition, even for constitutively activated Ret mutants, to rescue neuroectodermic cells from apoptosis. These findings may help clarify how a gain-of-function mutation can be associated with a developmental defect.

ACKNOWLEDGMENTS

We gratefully acknowledge Genentech for providing recombinant GDNF. We are indebted to E. Van Obberghen and N. Rochet for critical reading of the manuscript and to A. Grima, C. Serres-Ordonez and R. Grattery for illustration work.

This study is supported by funds from the Institut National de la Santé et de la Recherche Médicale (INSERM), Biomed (grant CEE BMH4 CT97-2107), the Association pour la Recherche contre le Cancer (grant 9872), La Ligue contre le Cancer, and the Associazione Italiana per la Ricerca sul Cancro. B.M. is a recipient of a postdoctoral fellowship from Biomed; R.B. was a postdoctoral fellow of INSERM (Poste vert) and is supported by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro (FIRC).