Abstract
Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.
Highlights
Noonan syndrome (NS) is a congenital genetic disorder causing cardiac and developmental defects[1,2]
As we have reported previously for wild type (WT) SOS23, single molecules of SOS were observed on the plasma membranes of the cells before and after stimulation with epidermal growth factor (EGF) (Fig. 1c)
After the cells were stimulated with a saturating concentration of EGF (100 ng/ml), the densities of the NS SOS molecules on the cell surfaces were increased (Fig. 1d)
Summary
Noonan syndrome (NS) is a congenital genetic disorder causing cardiac and developmental defects[1,2]. When SOS takes the open form and RAS–GTP binds to the REM domain, the GEF activity of SOS in the Cdc[25] domain is strongly increased[15,18], forming an SOS–RAS positive feedback loop. Some mutations in the H and PH domains are thought to increase the affinity of SOS for the lipid components of the membrane, and others are thought to destabilize the closed conformation, as suggested from the crystal structure[15]. These possibilities have not been examined experimentally in the context of living cells, and the molecular mechanisms by which some mutations cause NS are largely unknown
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
