Calcium depletion dissociates and activates heterodimeric notch receptors
Calcium depletion dissociates and activates heterodimeric notch receptors
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DOI：
10.1128/mcb.20.5.1825-1835.2000
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发表时间：
2000-03-01
影响因子：
5.3
通讯作者：
Aster, JC
中科院分区：
文献类型：
--
作者：
Rand, MD;Grimm, LM;Aster, JC
Notch受体参与调节多细胞动物形态发生的高度保守的信号通路。Notch受体的成熟需要单个前体多肽的蛋白酶解切割，以产生由配体结合胞外域（N-EC）和单程跨膜信号结构域（N-TM）组成的异二聚体。Notch信号传导与另外的配体诱导的蛋白水解裂解以及N-TM（N-ICD）的细胞内部分的核转位相关。在目前的工作中，我们表明，果蝇Notch和人Notch 1（hN 1）的NEC和N-TM亚基非共价相互作用。N-EC-N-TM相互作用被0.1%十二烷基硫酸钠或二价阳离子螯合剂如EDTA破坏，并被毫摩尔Ca 2+稳定。从N-EC亚基中删除Ca 2+结合Lin 12-Notch（LN）重复序列导致N-EC自发脱落到条件培养基中，这意味着LN重复序列在维持N-EC和N-TM的相互作用中是重要的。EDTA诱导的NEC解离的功能后果进行了研究，通过使用hN 1表达NIH 3 T3细胞。用0.5至10 mM EDTA处理这些细胞10至15 min导致N-EC快速脱落，N-ICD预期大小的多肽的瞬时出现，核内抗Notch 1染色增加，以及Notch敏感报告基因的瞬时激活。EDTA处理的HeLa细胞表达内源性Notch 1也刺激报告基因活性的程度相当于从暴露的细胞配体Delta 1。这些研究结果表明，受体活化可以发生作为N-EC解离的结果，这缓解了抑制的内在活性N-TM亚基。
Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N-EC) and a single-pass transmembrane signaling domain (N-TM). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N-TM (N-ICD). In the current work we show that the NEC and N-TM subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N-EC-N-TM interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca2+. Deletion of the Ca2+-binding Lin12-Notch (LN) repeats from the N-EC subunit resulted in spontaneous shedding of N-EC into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N-EC and N-TM. The functional consequences of EDTA-induced NEC dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N-EC, the transient appearance of a polypeptide of the expected size of N-ICD, increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N-EC dissociation, which relieves inhibition of the intrinsically active N-TM subunit.