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 alpha1-antitrypsin variant, alpha 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.

Figure 1

(A) The p120 processing product of Notch1, but not the full-length p300 precursor, is present at the cell surface. Jurkat T cells were labeled for 2 hr with [³⁵S]methionine and incubated with sulfo-NHS-LS biotin (+) or mock-treated (−). After lysis, extracts were immunoprecipitated with an antiserum raised against the intracellular region of Notch1 (antiserum IC) either directly (T) or after adsorption on streptavidin-agarose (B), and run on a 6.5% SDS-polyacrylamide gel. Positions of full-length Notch (p300) and p120 processing products are indicated. (B) Notch1 p120 is associated with the p200 N-terminal region of the molecule. Extracts from untransfected HeLa cells (lanes 3 and 4) or from HeLa cells stably transfected with murine Notch1 cDNA (lanes 1, 2, 5, and 6) were immunoprecipitated with the antiserum IC described above (lanes 1, 3, and 5) or with an irrelevant serum (lanes 2, 4, and 6) in TNT buffer. The precipitates were resolved on a 6.5% SDS-polyacrylamide gel, transferred onto Immobilon membranes, and probed with an antiserum raised against amino acids 1621–1637, located in the extracellular region (EC: lanes 1–4), or with serum IC (lanes 5 and 6). Molecular mass markers (kDa) are indicated on the left and the positions of p300, p200 (also marked by a square dot on the left), and p120 are shown on the right. ns, nonspecific band.

Figure 2

Inhibition of Notch1 receptor processing in the presence of BFA or A23187 in HeLa(N1) (A) and in the presence of α1-PDX in Jurkat cells (B). Identical amounts (100 μg) of the following extracts were run on a 6.5% SDS-polyacrylamide gel and incubated after transfer with antiserum IC. (A) Extracts from HeLa(N1) cells treated (3 hr) with BFA (50 μg/ml) (lanes 3 and 4) or with A23187 (2 μM) (lanes 1 and 2) in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of 50 μg/ml cycloheximide. (B) Extracts from Jurkat cells (lane 2) or Jurkat cells stably transfected with the α1-PDX cDNA (clone C12D, lane 1). Positions of full-length Notch (p300) and p120 processing product are indicated.

Figure 3

Notch1 processing does not take place in LoVo cells. HeLa cells (lanes 1–4) or LoVo cells, either transfected with Notch1 cDNA (lanes 5–8) or cotransfected with Notch1 and furin cDNAs (lanes 9–12) were labeled for 15 min with [³⁵S]methionine and then chased for the indicated times. Extracts were prepared and immunoprecipitated with antiserum IC and analyzed on 6.5% SDS-polyacrylamide gels. Molecular mass markers (kDa) are indicated on the left of lane 5, and the positions of p300 and p120 are shown.

Figure 4

Mutational analysis of Notch digestion by furin in vitro. (A) Schematic map of the recombinant substrate: murine Notch1 amino acids coordinates are indicated below the map. The main processing site (RQRR) and the two potential secondary sites are shown above the map, and the positions where processing by furin takes place are shown below. (B) The wild-type (WT, lanes 2 and 3) or the mutated (RQRR → AAAA; Mut, lanes 4 and 5) recombinant substrates were either mock-digested (lanes 3 and 4) or digested with purified recombinant BCRD-furin (lanes 2 and 5), and the products were analyzed on 15% Tricine gels followed by silver staining. The full-length substrate and N-terminal digestion products are indicated by dots on the left, and the C-terminal digestion products are indicated by arrows. The digestion product, purified on nickel-agarose, used for N-terminal sequencing is shown in lane 1. Molecular mass markers (kDa) are indicated on the right.

Figure 5

Processing of mutated Notch molecules transfected into 293T cells. Extracts from 293T cells either untransfected (lane 1) or transfected with full-length Notch1 cDNA (wt, lanes 2 and 6), mutated Notch1 cDNA (m1, mutation RQRR → AAAA; lanes 3 and 7), and Notch1 cDNA carrying the three mutations described in the text (m3 and m′3: lanes 4 and 5 and 8 and 9) in the absence (lanes 2–5) or presence (lanes 6–9) of cotransfected furin cDNA were analyzed by immunoblotting by using the IC antiserum. Positions of full-length Notch (p300) and p120 processing products are indicated. m3 and m′3 correspond to two independent experiments by using the Notch1 triple mutant.