A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling

Notch receptors are single transmembrane receptors that contain a large number of epidermal growth factor-like repeats (EGF repeats) in their extracellular domains. Mutations in the EGF repeats of the human Notch 3 receptor lead to the vascular dementia disease Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). The vast majority of CADASIL mutations are missense mutations removing or inserting cysteine residues in the EGF repeats, but it is not yet clear whether these mutations primarily affect receptor trafficking, maturation, and/or signaling. To address this issue, we have generated and analyzed stable cell lines expressing either wild-type murine Notch 3 (mNotch 3) or the mutant mNotch 3R142C, which corresponds to the prevalent CADASIL form of Notch 3, Notch 3R141C in humans. We find that a lower proportion of mNotch 3R142C is expressed in the site 1-cleaved configuration, and that reduced amounts of mNotch 3R142C appear at the cell surface, as compared with wild-type mNotch 3. This observation is accompanied by a higher propensity for mNotch 3R142C to form intracellular aggregates, which may be a result of increased accumulation or slowed transport in the secretory pathway. In contrast to the impaired cell surface expression, mNotch 3R142C signals equally well in response to Delta 1 and Jagged 1 as wild-type mNotch 3. Taken together, these data suggest that trafficking and localization rather than signaling of mNotch 3 are affected in mNotch 3R142C.

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