CADASIL associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 receptor signaling

Mutations in the Notch3 gene are the cause of CADASIL, a hereditary microangiopathy leading to stroke and vascular dementia. Typical mutations lead to an odd number of cysteine residues within an epidermal-growth-factor (EGF-) repeat in the extracellular domain of the Notch3 transmembrane receptor. In CADASIL, the extracellular domain of Notch3 (N3ECD) is deposited in the vicinity of vascular smooth muscle cells (VSMC), which have been shown to degenerate in patients. However, the mechanisms underlying VSMC degeneration and N3ECD accumulation are still unknown.

Here we investigated the consequences of three pathogenic Notch3 mutations (R133C, C183R, C455R) on ligand- (Delta1-) binding to the receptor and on the biological activity of the receptor by analysing ligand- (Jagged-) induced signaling via RBP-Jk in NIH3T3 cells as well as VSMCs (A7r5). All mutant receptors were targeted to the cell surface. However, two mutations (R133C and C183R) which are located outside the putative ligand binding domain (LBD) of the receptor were found to result in normal Jagged1-induced signaling in A7r5 VSMC whereas the third mutation (C455R located within the putative LBD) showed strongly reduced signaling acitivity. Ligand binding assays with soluble Delta1Fc revealed that C455R interferes with ligand binding through disruption of the LBD which, as we show here, is located in EGF repeats 10/11 of Notch3. Taken together, these data indicate that CADASIL associated Notch3 mutations differ with respect to their consequences both on ligand binding and ligand-induced signaling through RBP-Jk. They further suggest, that ligand-induced receptor shedding may not be required for the deposition of Notch3 extracellular domain in CADASIL.