Calcium depletion dissociates and activates heterodimeric notch receptors

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.