title: CADASIL-associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 receptor signaling through RBP-Jk.
abstract: Mutations in the NOTCH3 gene are the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary angiopathy leading to strokes and dementia. Pathogenic mutations remove or insert cysteine residues within epidermal growth factor (EGF) repeats in the extracellular domain of the Notch3 receptor (N3ECD). Vascular smooth muscle cells (VSMC) are the predominant site of Notch3 expression in adults. In CADASIL patients, VSMC degenerate and N3ECD is deposited within the vasculature. However, the mechanisms underlying VSMC degeneration and N3ECD accumulation are still unknown. In this study, we investigated the consequences of three pathogenic Notch3 mutations on the biological activity of the receptor by analyzing ligand (Delta-/Jagged-)-induced signaling via RBP-Jk. Two mutations (R133C and C183R) that 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 activity. Ligand binding assays with soluble Delta1 and Jagged1 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. All mutant receptors including Notch3C455R were targeted to the cell surface but showed an elevated ratio between the unprocessed full-length 280-kDa receptor and S1-cleaved receptor fragments. 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, whereas they have similar effects on receptor maturation. Moreover, the data suggest that ligand-induced receptor shedding may not be required for N3ECD deposition in CADASIL.
authors.affiliation: Department of Neurology, Klinikum Grosshadern, Ludwig-Maximilians University, Munich, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Peters
authors.forename: Christian
authors.initials: C
authors.lastname: Opherk
authors.forename: Simone
authors.initials: S
authors.lastname: Zacherle
authors.forename: Anja
authors.initials: A
authors.lastname: Capell
authors.forename: Petra
authors.initials: P
authors.lastname: Gempel
authors.forename: Martin
authors.initials: M
authors.lastname: Dichgans
mesh_terms: D000328:Adult; D000818:Animals; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015161:Dementia, Multi-Infarct / Q000235:genetics*; D004815:Epidermal Growth Factor / Q000737:chemistry; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D036341:Intercellular Signaling Peptides and Proteins; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins; D051379:Mice; D009154:Mutation / Q000235:genetics*; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D000071656:Receptor, Notch3; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D011971:Receptors, Immunologic / Q000378:metabolism*; D051880:Receptors, Notch; D045522:Recombinases / Q000378:metabolism; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction
chemical_list: D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; C000606274:Jag1 protein, mouse; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins; C499374:NOTCH3 protein, human; C499373:Notch3 protein, mouse; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; C497570:RBPJ protein, human; C497571:Rbpj protein, mouse; D000071656:Receptor, Notch3; D000074562:Receptor, Notch4; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D051880:Receptors, Notch; D045522:Recombinases; D000072099:Serrate-Jagged Proteins; C073572:Notch4 protein, mouse; D004815:Epidermal Growth Factor; D003513:Cycloheximide