THE NOTCH1 RECEPTOR IS CLEAVED CONSTITUTIVELY BY A FURIN-LIKE CONVERTASE

The Notch receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precurso r molecule (p300), We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active recepto r because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notc h receptor exists at the plasma membrane as a heterodimeric molecule, but disagree as to the nature of the protease that is responsible for the cleavage that takes place in the extracellular region. We report h ere that constitutive processing of murine Notch1 involves a furin-lik e convertase. We show that the calcium ionophore A23187 and the alpha 1-antitrypsin variant, alpha 1-PDX, a known inhibitor of furin-like co nvertases, inhibit p300 processing. When expressed in the furin-defici ent Love cell line, p300 is not processed. In vitro digestion of a rec ombinant Notch-derived substrate with purified furin allowed mapping o f the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654), Mutation of these four amino acids (and of two seco ndary dibasic furin sites located nearby) completely abolished process ing of the Notch1 receptor.