We further verified the binding of the Crb extracellular domains to Notch1a using a cell-surface binding assay. We constructed the plasmids encoding the extracellular domains of the Crb family proteins fused to the Fc portion of human IgG to generate soluble forms (Crb-Fc) and prepared conditioned media from cultures of 293T cells that were transfected with these vectors. We then added the conditioned media that contained the Crb-Fc fusion proteins to nonpermeabilized 293T cells that were transfected with a Notch1a PI3K cancer construct in which the intracellular ankyrin repeats and the transactivation domain were replaced with EYFP (hereafter referred to as EcRAM-EYFP since

it consists of the extracellular domain and the intracellular RBP-J association module) ( Eiraku et al., 2005) ( Figure 5Ba). Crb-Fc specifically bound to the surfaces of the EcRAM-EYFP-expressing cells but not to the surfaces of EcRAM-EYFP-nonexpressing cells (dotted lines in Figures 5Bb–5Bp). Vitamin D binding protein (VDBP) fused with the Fc portion of human IgG did not bind to either EcRAM-EYFP-expressing or EcRAM-EYFP-nonexpressing cells ( Figures 5Bf, 5Bk, and 5Bp). These results indicate that the extracellular domains of the Crb family proteins specifically and directly interact with the Notch1a extracellular domain. Next, we examined the effects of the Crb family proteins on Notch activity using luciferase reporter assays and the C2C12

myoblast cell line (Shawber et al., 1996). We transfected C2C12 cells with the Notch-responsive reporter construct (pGa981-6) (Kurooka et al., 1998). When the C2C12 cells were cocultured selleck kinase inhibitor with mock-transfected 293T cells, the promoter activity was increased approximately 6.5-fold compared with that of monocultured C2C12 cells, presumably because of the presence of endogenous Notch ligands in the 293T cells (Figure 5C, columns 1 and 2). This activation was further enhanced more than 12-fold by the overexpression Sclareol of murine Delta-like 1 (Dll1, formerly known as Delta1) in the 293T cells (Figure 5C, column 3). However, the incubation of Crb-Fc with the C2C12 cells

prior to coculturing with Dll1-overexpressing 293T cells reduced Notch activity to the basal level (Figure 5C, columns 4–6). In addition, overexpression of Crb family proteins in C2C12 cells reduced the Notch activity (Figure 5C, columns 7–9). In contrast, the Crb family proteins did not affect the activation of Notch when coexpressed with Dll1 in 293T cells (p = 0.34) (Figures 5Da and 5Db), which suggests that the Crb inhibition of Notch signaling in the present study occurs mainly in cis. We further examined the effect of Moe on the inhibition of Notch activity by Crb using CaCo-2 cells, which are human epithelial colorectal adenocarcinoma cells with apicobasal polarity. In CaCo-2 cells, Notch was also activated by coculturing with Dll1-expressing 293T cells, and this activation was inhibited by the expression of Crb2 in the CaCo-2 cells (Figures 5Ea and 5Eb).