Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors

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Abstract
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 (NEC) and a single-pass transmembrane signaling domain (NTM). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of NTM(NICD). In the current work, we show that the NEC and NTM subunits of DrosophilaNotch and human Notch1 (hN1) interact noncovalently. NEC-NTM 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 NEC subunit resulted in spontaneous shedding of NEC into conditioned medium, implying that the LN repeats are important in maintaining the interaction of NEC and NTM. 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 NEC, the transient appearance of a polypeptide of the expected size of NICD, 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 NEC dissociation, which relieves inhibition of the intrinsically active NTMsubunit.