title: Functional analysis of a recurrent missense mutation in Notch3 in CADASIL.
abstract: BACKGROUND
  Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterised by recurrent ischemic strokes in the deep white matter. Mutations in the gene encoding the cell surface receptor, Notch3, have been identified in CADASIL patients, and accumulation of the extracellular domain of Notch3 has been demonstrated in affected vessels. Almost all CADASIL mutations alter the number of cysteine residues in the epidermal growth factor (EGF)-like repeats in the extracellular domain of the protein.
  
  
  OBJECTIVE
  To understand the functional consequences of a recurrent CADASIL mutation on furin processing, cell surface expression, ligand binding, and activation of a downstream effector CBF1 by the Notch3 receptor.
  
  
  METHODS
  We expressed wild type and mutant Notch3 receptors in cultured cells and examined cell surface expression of the proteins. We also applied a new flow cytometry based approach to semi-quantitatively measure binding to three Notch ligands. Additionally, we used a well characterised co-culture system to examine ligand dependent activation of transcription from a CBF1-luciferase reporter construct.
  
  
  RESULTS
  These studies revealed subtle abnormalities in furin processing of the mutant receptor, although both heterodimeric and full length receptors are present on the cell surface, are capable of interacting with soluble forms of three ligands, Delta1, Delta4, and Jagged1, and retain the ability to activate CBF1 in a ligand dependent manner.
  
  
  CONCLUSIONS
  By comparison with other mutant forms of Notch3, these data indicate that individual CADASIL mutations can have disparate effects on Notch3 expression and function.
authors.affiliation: Nemours Biomedical Research, Alfred I duPont Hospital for Children, 1600 Rockland Road, Wilmington, DE 19803, USA.
authors.forename: T
authors.initials: T
authors.lastname: Haritunians
authors.forename: T
authors.initials: T
authors.lastname: Chow
authors.forename: R P J
authors.initials: RP
authors.lastname: De Lange
authors.forename: J T
authors.initials: JT
authors.lastname: Nichols
authors.forename: D
authors.initials: D
authors.lastname: Ghavimi
authors.forename: N
authors.initials: N
authors.lastname: Dorrani
authors.forename: D M
authors.initials: DM
authors.lastname: St Clair
authors.forename: G
authors.initials: G
authors.lastname: Weinmaster
authors.forename: C
authors.initials: C
authors.lastname: Schanen
mesh_terms: D000328:Adult; D046589:CADASIL / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000502:physiology; D005434:Flow Cytometry; D045683:Furin / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008024:Ligands; D008297:Male; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000502:physiology; D010375:Pedigree; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D000071656:Receptor, Notch3; D011956:Receptors, Cell Surface / Q000235:genetics*; D051880:Receptors, Notch
chemical_list: D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008024:Ligands; C499374:NOTCH3 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497570:RBPJ protein, human; D000071656:Receptor, Notch3; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D045683:Furin