Calcium Depletion Dissociates and Activates Heterodimeric Notch Receptors

Abstract

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 (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 N EC 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 Ca 2+ . Deletion of the Ca 2+ -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 N EC 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.