Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and 
Leukoencephalopathy (CADASIL) is the best understood cause of dominantly 
inherited stroke and results from NOTCH3 mutations that lead to NOTCH3 protein 
accumulation and selective arterial smooth muscle degeneration. Previous studies 
show that NOTCH3 protein forms multimers. Here, we investigate protein 
interactions between NOTCH3 and other vascular Notch isoforms and characterize 
the effects of elevated NOTCH3 on smooth muscle gene regulation. We demonstrate 
that NOTCH3 forms heterodimers with NOTCH1, NOTCH3, and NOTCH4. R90C and C49Y 
mutant NOTCH3 form complexes which are more resistant to detergents than wild 
type NOTCH3 complexes. Using quantitative NOTCH3-luciferase clearance assays, we 
found significant inhibition of mutant NOTCH3 clearance. In coculture assays of 
NOTCH function, overexpressed wild type and mutant NOTCH3 significantly 
repressed NOTCH-regulated smooth muscle transcripts and potently impaired the 
activity of three independent smooth muscle promoters. Wildtype and R90C 
recombinant NOTCH3 proteins applied to cell cultures also blocked canonical 
Notch fuction. We conclude that CADASIL mutants of NOTCH3 complex with NOTCH1, 
3, and 4, slow NOTCH3 clearance, and that overexpressed wild type and mutant 
NOTCH3 protein interfere with key NOTCH-mediated functions in smooth muscle 
cells.