The Notch1 receptor is cleaved constitutively by a furin-like convertase

Abstract

The Notch receptor, which is involved in numerous cell fate decisions in invertebrates and vertebrates, is synthesized as a 300-kDa precursor molecule (p300). We show here that proteolytic processing of p300 is an essential step in the formation of the biologically active receptor because only the cleaved fragments are present at the cell surface. Our results confirm and extend recent reports indicating that the Notch 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 here that constitutive processing of murine Notch1 involves a furin-like convertase. We show that the calcium ionophore A23187 and the ␣1-antitrypsin variant, ␣ 1-PDX, a known inhibitor of furin-like convertases, inhibit p300 processing. When expressed in the furin-deficient Lovo cell line, p300 is not processed. In vitro digestion of a recombinant Notch-derived substrate with purified furin allowed mapping of the processing site to the carboxyl side of the sequence RQRR (amino acids 1651-1654). Mutation of these four amino acids (and of two secondary dibasic furin sites located nearby) completely abolished processing of the Notch1 receptor.

Identification of a novel NOTCH-4/INT-3 RNA species encoding an activated gene product in certain human tumor cell lines

“…As shown in Figure 5b (lanes 2 ± 6), multiple bands were detected in protein extracts of the di erent cell lines. The multiple bands observed in Figure 5b undoubledly re¯ect, in part, the previously noted complex array of proteolytic cleavage products that accumulate during maturation of NOTCH proteins De Strooper et al, 1999;Hardy et al, 1999;Logeat et al, 1998;Struhl et al, 1999;Ye et al, 1999). The speci®city of this reactivity was demonstrated by the ability of the immunogenic peptide to block the reaction (Figure 5c).…”

High delta‐like 1 expression in a subset of neuroblastoma cell lines corresponds to a differentiated chromaffin cell type

“…Therefore, Notch receptors are presented on the cell surface as intra-molecular heterodimers. 32 The Notch1 and Notch2 antibodies detect the 110 kDa C-terminus of these Notch proteins (Fig. 2c,d).…”

Does Notch play a tumor suppressor role across diverse squamous cell carcinomas?

“…The large Notch extracellular domain (NECD) comprises 11–36 tandemly repeated epidermal growth factor‐like (EGF) repeats followed by a negative regulatory region (NRR) composed of three Lin12‐Notch repeats (LNR) and a hetero‐dimerization domain (HD). The HD domain is cleaved at S1 site by a furin‐like protease within the secretory pathway 12, generating a heterodimer that remains in a resting state, with its NRR domain being resistant to further protease cleavage 13, 14.…”