Structure-based mechanistic principles for the paradoxical effects of pathological RET mutations

Abstract

The RET receptor is a transmembrane protein that belongs to the receptor tyrosine kinase family. RET activates signaling pathways regulating cell growth, differentiation, and survival in diverse tissues that include the thyroid and enteric nervous system. Mutations that reduce RET levels or disrupt function can cause Hirschsprungs disease (HSCR), characterized by abnormal distal colon innervation in the developing embryo. In contrast, mutations that constitutively activate RET can lead to tumorigenesis, most notably in multiple endocrine neoplasia type 2 (MEN2) syndromes usually involving the thyroid. Paradoxically, some RET mutations can both reduce RET activity in enteric ganglia, and increase signaling in other tissues, resulting in patients suffering from both HSCR and MEN2A. Although extensive research has been conducted on RET mutations, the structural and mechanistic bases underlying these paradoxical effects remain unclear. Here, we curated data on 70 positions in RET extracellular domains where point-mutations were associated with HSCR, MEN2A, or both. Taking a structure-based approach, we predict the potential effects of mutations in these positions on RET structure. Our analysis suggests that approximately 90% of positions associated with HSCR can, upon mutation, disrupt intramolecular interactions that stabilize RET tertiary structure: residues buried in the protein core, calcium-binding sites, or residues participating in stabilizing intramolecular electrostatic/covalent bonds. A smaller subset of mutations involves substitutions to/from glycines or prolines in key positions. Only a small minority of HSCR-associated positions affect protein-protein interactions needed for signal activation. On the other hand, our analysis showed that [~]75% of mutations in positions that cause MEN2A lead to an unpaired cysteine that can form an intermolecular disulfide bond between two RET monomers. Other mutations that cause MEN2A are also predicted to enhance RET homodimerization via extracellular domains that are proximal to the membrane. Importantly, substitutions that concurrently destabilizes RET tertiary structure and lead to an unpaired cysteine are predicted to cause the paradoxical co-occurrence of HSCR and MEN2A. Our findings suggest a mechanistic basis for almost all identified pathological mutations in RET and imply that therapeutic strategies for targeting RET activity in HSCR and MEN2A may need to be orthogonal.