Joutel Anne, Monet Marie, Domenga Valérie, Riant Florence, Tournier-Lasserve Elisabeth
Institut National de la Santé et de la Recherche Médicale, INSERM E365, Faculté de Médecine Lariboisière, and Laboratoire de Cytogénétique, Hôpital Lariboisière, Paris, France.
Am J Hum Genet. 2004 Feb;74(2):338-47. doi: 10.1086/381506. Epub 2004 Jan 8.
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterized by the degeneration of smooth-muscle cells in small cerebral arteries. CADASIL is caused by mutations in NOTCH3, one of the four mammalian homologs to the Drosophila melanogaster NOTCH gene. Disease-associated mutations are distributed throughout the 34 epidermal growth factor-like repeats (EGFRs) that compose the extracellular domain of the Notch3 receptor and result in a loss or a gain of a cysteine residue in one of these EGFRs. In human adults, Notch3 expression is highly restricted to vascular smooth-muscle cells. In patients with CADASIL, there is an abnormal accumulation of Notch3 in the vessel. Molecular pathways linking NOTCH3 mutations to degeneration of vascular smooth-muscle cells are as yet poorly understood. In this study, we investigated the effect of CADASIL mutations on Notch3 activity. We studied five naturally occurring mutations: R90C and C212S, located in the previously identified mutational hotspot EGFR2-5; C428S, shown in this study to be located in the ligand-binding domain EGFR10-11; and C542Y and R1006C, located in EGFR13 and EGFR26, respectively. All five mutant proteins were correctly processed. The C428S and C542Y mutant receptors exhibited a significant reduction in Jagged1-induced transcriptional activity of a RBP/JK responsive luciferase reporter, relative to wild-type Notch3. Impaired signaling activity of these two mutants arose through different mechanisms; the C428S mutant lost its Jagged1-binding ability, whereas C542Y retained it but exhibited an impaired presentation to the cell surface. In contrast, the R90C, C212S, and R1006C mutants retained the ability to bind Jagged1 and were associated with apparently normal levels of signaling activity. We conclude that mutations in Notch3 differently affect Jagged1 binding and Notch3 signaling via the RBP/JK pathway.
伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病（CADASIL）是一种遗传性血管性痴呆，其特征为大脑小动脉平滑肌细胞变性。CADASIL由NOTCH3突变引起，NOTCH3是果蝇NOTCH基因在哺乳动物中的四个同源基因之一。与疾病相关的突变分布在构成Notch3受体细胞外结构域的34个表皮生长因子样重复序列（EGFR）中，并导致其中一个EGFR中半胱氨酸残基的缺失或增加。在成年人体内，Notch3的表达高度局限于血管平滑肌细胞。在CADASIL患者中，血管内存在Notch3异常积聚。将NOTCH3突变与血管平滑肌细胞变性联系起来的分子途径目前仍知之甚少。在本研究中，我们调查了CADASIL突变对Notch3活性的影响。我们研究了五个自然发生的突变：R90C和C212S，位于先前确定的突变热点EGFR2 - 5；C428S，本研究表明其位于配体结合结构域EGFR10 - 11；以及C542Y和R1006C，分别位于EGFR13和EGFR26。所有五个突变蛋白均得到正确加工。相对于野生型Notch3，C428S和C542Y突变受体在Jagged1诱导的RBP/JK反应性荧光素酶报告基因的转录活性方面表现出显著降低。这两个突变体信号活性受损是通过不同机制产生的；C428S突变体失去了其与Jagged1结合的能力，而C542Y保留了该能力，但在细胞表面的呈现受损。相比之下，R90C、C212S和R1006C突变体保留了与Jagged1结合的能力，并与明显正常水平的信号活性相关。我们得出结论，Notch3中的突变通过RBP/JK途径对Jagged1结合和Notch3信号传导有不同影响。
