Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1

Abstract

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

Figure 1.

CR-1 physically associates with all four Notch receptors. (A–D) Flag-tagged CR-1 (CR-Flag) was cotransfected with HA-tagged Notch1 (N1FL-HA; A), Myc-tagged Notch2 (N2FL-Myc; B), HA-tagged Notch3 (N3FL-HA; C), and V5-tagged Notch4 (N4FL-V5; D) in COS-7 cells. IP and immunoblotting (IB) were performed with the indicated antibodies. cl., cleaved Notch proteins; fl., FL Notch proteins. (E) Nonquantitative RT-PCR for Notch receptor expression in NTERA2/D1 cells. M, markers. (F and G) Interaction between endogenous CR-1 and Notch1/2 in NTERA2/D1 cells. (F) Notch1 IP was performed using anti-Notch1 polyclonal antibodies (C20 and AF5317). (G) CR-1 IP was performed with anti–CR-1 goat polyclonal antibody (α–CR-1). Normal goat or sheep IgGs were used as negative controls. Proteins were detected with the indicated antibodies. (H) Flag-tagged CFC1 (CFC-Flag) was cotransfected with N1FL-HA, and co-IP was performed as described in A–D.

Figure 2.

Deletion analysis of the CR-1–Notch interaction. (A) CR-1 deletions. (B) Notch1 deletions/chimeras. (C–F) Co-IP was performed using anti-Flag (C), anti-HA (D and E), or anti-Myc (F) affinity beads. Proteins were detected with the indicated antibodies. cl., cleaved Notch proteins; EV, empty vector; fl., FL Notch proteins; IB, immunoblot; S.S., signal sequence.

Figure 3.

Intracellular interaction of CR-1 and Notch1. (A) Cell surface biotinylation assay. Transiently transfected COS-7 cells were treated with N-hydroxysuccinimide–PEG₄-biotin. Co-IP or sequential co-IP was performed with the indicated antibodies. Each indicated band corresponds as follows: 1, biotinylated CR-1; 2, unbiotinylated and glycosylated CR-1; 3, unbiotinylated and unglycosylated CR-1; 4, carryover of 3× Flag peptides used for Flag elution. (B) Effect of glycosylation on the CR-1–Notch1 interaction. COS-7 cells were treated with vehicle or 10 µg/ml tunicamycin for 16 h after transient transfection. Co-IP assays were performed reciprocally. (C) Intracellular localization of CR-1 and Notch1. GFP-tagged CR-1 and N1FL-HA were visualized by a confocal microscope. (D) Cell surface expression of CR-1 after BFA treatment. Transiently transfected COS-7 cells were treated with the indicated concentrations of BFA for 16 h. Cells were stained with PE-conjugated anti–CR-1 mAb, and FACS analysis was performed. (E) Transiently transfected COS-7 cells were treated with vehicle or 2 µg/ml BFA for 16 h, and the co-IP experiment was performed using the indicated antibodies. cl., cleaved Notch proteins; fl., FL Notch proteins; IB, immunoblot.

Figure 4.

Sensitization of the Notch signaling pathway by CR-1. (A) TP-1 reporter assay of co-cultured CHO cells with L-WT, L-Dll1, or L–Jagged-1 cells. CHO cells were transiently transfected with empty vector (EV) or WT CR-1 expression vector before co-culture. Cotransfection of FL Notch1 (N1FL) was also performed. Mean ± SD is shown for three independent experiments. *, P < 0.05. (B) Sucrose gradient isolation of lipid rafts in transiently transfected CHO cells. Fractions 4–5 correspond to the lipid raft fractions. Transferrin receptor (TfR) or Cholera toxin B (CTxB) was used as a nonraft or lipid raft marker, respectively. IB, immunoblot. (C) S1 cleavage sites of Notch1. ANK, ankyrin domain; S.S., signal sequence; TM, transmembrane domain. (D and E) Enhancement of S1 cleavage of Notch1 by CR-1 expression. CHO cells transiently transfected with the indicated amount of expression vectors were incubated with 10 µM DAPT for 24 h and analyzed by Western blotting. Mean ± SD of densitometric quantification is shown for three independent transfections (E). (F) Blockade of CR-1–induced Notch processing by a furin inhibitor. Transiently transfected CHO cells were treated with the indicated concentrations of a furin inhibitor, Decanoyl-RVKR-chloromethylketone, for 24 h, and Notch processing was analyzed as described in D. (G) Enhanced cell surface expression of Notch1 by CR-1. CHO cells were transfected with the indicated expression vectors, and the cell surface expression level of Notch1 was assessed by FACS analysis. Arrows indicate the peaks of Notch1-transfected populations.

Figure 5.

Functional interaction between endogenous CR-1 and Notch1 in EC cells. (A–C) Endogenous expression of CR-1 and Notch receptors in F9 WT and Cr^−/− cells. (A) Nonquantitative RT-PCR. Samples treated without reverse transcription (RT−) were used as negative controls. (B) Quantitative RT-PCR. Mean ± SD is shown for four independent cultures. *, P < 0.05 compared with F9 WT. (C) Western blot analysis. Empty vector– and N1FL-transfected CHO cells (CHO EV and N1FL) were used as negative and positive controls, respectively. cl., cleaved Notch proteins; fl., FL Notch proteins. (D and E) FACS analysis for the cell surface expression of endogenous Notch1 in F9 cells. Mean ± SD is shown for three independent cultures (E). *, P = 0.021. (F) Effect of Cr-1 knockdown on ligand-induced TP-1 reporter activity in F9 cells. Transiently transfected F9 WT or Cr^−/− cells were co-cultured with L-WT, L-Dll1, or L–Jagged-1 cells. Cotransfection of N1FL was also performed. Mean ± SD of relative values of relative luciferase units is shown for four independent experiments. *, P < 0.05. (G and H) siRNA knockdown of CR-1 in NTERA2/D1 cells. Suppression of CR-1 protein by siCR-1_1 and siCR-1_2 was evaluated by Western blotting (G) and FACS analysis (H, left). GFP was used as an indicator of transfection efficiency (>95%; H, right). (I) Effect of CR-1 knockdown on ligand-induced Notch target gene expression in NTERA2/D1 cells. siRNA-transfected NTERA2/D1 cells were co-cultured with L-WT, L–Dll1, or L–Jagged-1 cells. Human Notch target gene expression was assessed by quantitative RT-PCR. Mean ± SD of relative values is shown for four independent experiments. *, P < 0.05.