Notch responds differently to Delta and Wingless in cultured Drosophila cells.

Notch, a cell surface receptor, is required for producing different types of cells during development of Drosophila melanogaster. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to ligand Wingless. The means by which Notch initiates these different intracellular activities was examined in this study. Cultured cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta resulted in accumulation of approximately 120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch did not accumulate in cells treated with Wingless, but the approximately 350-kDa full-length Notch molecules accumulated. These results indicate that N responds differently to binding by Delta and Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane.

Notch (N) 1 is required for the specification of different cell types during development of Drosophila melanogaster (1). It is a cell surface receptor, the intracellular activities of which are regulated by ligands binding to the extracellular domain. Delta (Dl) is the ligand for the well known N functions associated with lateral inhibition. During lateral inhibition, N and Dl produce the neuronal precursor cells that differentiate the nervous system and the epidermal precursor cells that differentiate the cuticle (1)(2)(3)(4). Wingless is the ligand for some N functions associated with differentiation of the cuticle (5-10). The full-length N binds both Dl and Wg, in vivo and in vitro (7). It regulates expression of the Enhancer of split Complex and wingless in response to Dl (1,(11)(12)(13)(14). In response to Wg, it regulates expression of patched, shaggy, and hairy, but not Enhancer of split Complex and wingless (7). Thus, the same receptor regulates different sets of genes in response to Dl and Wg.
N intracellular signals in response to Dl are mediated by the Suppressor of Hairless (Su(H)) signal transduction pathway (11)(12)(13)(15)(16). The signaling pathway used by N to transduce the intracellular signals in response to Wg is unknown. Because Wg binding does not activate expression of Enhancer of split Complex and wingless (7), it is unlikely to be the same Su(H) pathway used with Dl. Furthermore, Wg-dependent functions of N during development are distinct from Dl-dependent N functions (5)(6)(7)(8)(9)(10). These observations indicate that fulllength N generates different intracellular signals in response to Dl and Wg. However, how can the same N receptor generate one signal after binding Dl and a different signal after binding Wg?
We treated N-expressing cultured cells with Dl and Wg and found out that N responds differently to binding by these two ligands. This differential response likely initiates transduction of different signals to the nucleus.

MATERIALS AND METHODS
S2-N and S2-Dl cells are Schneider (S2) cells transfected with the Notch and Delta genes, respectively, for expression of their proteins under the control of heat shock promoter (17). S2-N ⌬EGF1-18 and S2-N ⌬EGF19 -36 cells are S2 cells transfected with the Notch gene designed to express N proteins without epidermal growth factor-like (EGF-like) repeats 1 to 18 and EGF-like repeats 19 to 36, respectively. S2 cells do not express N, Dl, or Wg (4,7,18). Clone-8 are imaginal disc cells that express N and Dl, but not Wg (18). 2 S2 and Wg media were prepared by growing heat-shocked S2 cells or S2-Wg cells in Shields and Sang's M3 media as described (7).
1-2 ϫ 10 6 heat-shocked S2-N, S2-N ⌬EGF1-18 , S2-N ⌬EGF19 -36 , or clone-8 cells were treated with 1.5-3 ϫ 10 6 heat-shocked S2 cells or heat-shocked S2-Dl cells in the indicated media and incubated with gentle shaking at 25°C. Siliconized multiwell Falcon plates were used for incubation. Aliquots of the same cell solutions or media were used for each experiment. Proteins were extracted with 0.75% Triton X-100 and 0.5% deoxycholate as described (7). Subcellular fractions were prepared as described (16). Protein content in extracts was equalized by using absorbance values at 280 nm and the Bio-Rad DC protein assay kit. 8% SDS-polyacrylamide gel electrophoresis was used for electrophoresis. Western blotting was performed as described (19), and the signals were detected with an ECL kit (Amersham Pharmacia Biotech). ␣NI antibody, made against the intracellular CDC10/ankyrin region (20), was used to detect N molecules.

RESULTS
Schneider cells expressing Notch (S2-N cells) treated with Dl for 1 h accumulated ϳ120-kDa N molecules (N120; Fig. 1a, lanes 1 and 2). Dl binds N in the extracellular region including EGF-like repeats 11 and 12 (21). S2 cells expressing N molecules lacking this region, N ⌬EGF1-18 , do not accumulate N120 molecules in response to treatment with Dl ( Fig. 1a, lanes 3 and  4). This indicates that N120 accumulated in response to Dl binding N. N120 is the complete intracellular domain and is similar to the ϳ120-kDa N intracellular domain molecule shown to accumulate in vivo in response to Dl (1,16,22,23). 3 N120 molecules did not accumulate in S2-N cells treated with Wg for 1, 2, or 5 h (Fig. 1a, lanes 5 and 6, 9 and 10, and 12 and 13). However, S2-N cells treated with Wg for 5 h accumulated ϳ350-kDa N molecules (N350) but not S2-N cells treated with Dl (Fig. 1a, lanes [11][12][13]. N350 is the full-length co-linear N molecule containing both the intracellular and extracellular domains (7,16). Wg binds N in the EGF-like repeats 19 -36 region (7). S2 cells expressing N molecules lacking this region, N ⌬EGF19 -36 , did not accumulate co-linear molecules when treated with Wg for 5 h (Fig. 1a, lanes 15 and 16). On the other hand, truncated, co-linear N ⌬EGF1-18 molecules containing the Wg binding sites accumulated upon treatment with Wg ( Fig.  1a, lanes 17 and 18). These results indicate that accumulation of N350 in S2-N cells was in response to Wg binding N. Accumulation of N350 molecules was also discernible in cells treated with Wg for 2 h when the blots are exposed to film for shorter periods. In contrast to Wg-treated cells, Dl-treated cells in the same blots always had lower levels of N350 compared with the levels in untreated cells (data not shown, see below).
Accumulation of N350 molecules in Wg-treated cells is not due to activity of the endogenous Notch gene, which is rearranged in S2 cells (4). 2 It is not due to a general increase or stabilization of all proteins in the cells: all N molecules do not accumulate, and the total protein levels in the three lanes are comparable (see N120 and other molecules marked with an asterisk in Fig. 1a, lanes 11-13, and the HSP 70 panels). It is also not due to a Wg effect that is unrelated to N binding but retards N processing for cell surface presentation (see Refs. 24 -26 for cell surface N processing). Otherwise, co-linear N ⌬EGF19 -36 would have also accumulated, but it did not (see Fig. 1a, lanes 15 and 16). Thus, whereas Dl binding full-length N results in accumulation of N120, Wg binding results in accumulation of the co-linear N350.
Treatment of S2-N cells with Dl or Wg for 2 h also resulted in accumulation of ϳ55-kDa N molecules (N55; Fig. 1a, lanes   7-10). N55 contains only the amino terminus half of the intracellular domain, requires about 2 h to accumulate, and is variably recovered after about 3 h of treatment. 3 To determine whether the responses observed in S2 cells are general N responses to treatments with Dl and Wg, the experiments were repeated with clone-8 cells that express N endogenously (Fig. 1b). The results showed that N in clone-8 cells responded similarly to N in S2 cells. Treatment with Dl resulted in accumulation of N120 and not N350, whereas treatment with Wg resulted in accumulation of N350 and not N120; both Dl and Wg treatments resulted in accumulation of N55 molecules (Fig. 1b). The difference in levels of N350 between Dl-treated and Wg-treated cells is obvious here after just 2 h of treatment. Clone-8 cells express a higher level of N55 molecules in the absence of any treatment, presumably because they also express Dl endogenously. 2 When Dl binds N in vivo, the ϳ120-kDa N intracellular domain is released into the cytoplasm (1,15,16,22,23). To determine whether the N120 in our in vitro experiments with Dl also accumulated in the cytoplasm, S2-N cells were fractionated and analyzed following treatments with Dl and Wg. Following treatment with Dl, N120 molecules accumulated in the cytoplasmic fraction (Fig. 1c). In contrast, N350 molecules accumulated in the membrane fraction following treatment with Wg (Fig. 1c). N55 molecules are not consistently detected in these experiments as they are very unstable in this fractionation and extraction procedure (not shown).
We do not know whether the N120 molecules that accumulate in the cytoplasm in response to Dl are the same as those present in the membranes (see Fig. 1c) or whether they are different molecules migrating in the same region of the gel. Membrane-tethered N intracellular domain (N intra ), untethered N intra , and N120 migrate alongside each other in these gels. 2 N120 molecules associated with the membranes or with the cytoplasm are probably the membrane-tethered or released N intracellular domain, respectively. Accumulation of N350 molecules in response to Wg is likely to be in the intracellular membranes associated with production of the heterodimeric cell surface receptor (see Refs. 24 -26). N55 is derived from N350 upon activation of Notch signaling by a ligand. 3

DISCUSSION
In vivo, the complete N intracellular domain (ϳ120 kDa) is released from the plasma membrane in response to Dl. This domain translocates to the nucleus with Su(H) and activates expression of target genes (13, 15, 16, 22, 23). In our experiments, Dl treatment results in accumulation of ϳ120-kDa N intracellular domain molecules (N120) in the cytoplasm. N120  2 and 7 and 8) whereas S2-N cells treated with Wg accumulated the co-linear N350 and N55 molecules (lanes 5 and 6, 9 and 10, and 11-13). N120 molecules did not accumulate in S2-N ⌬EGF1-18 cells treated with Dl (lanes 3 and 4). Co-linear molecules also accumulated in S2-N ⌬EGF1-18 cells treated with Wg (lanes 17 and 18) but did not accumulate in N ⌬EGF19 -36 cells (lanes 15 and 16). S2-N cells treated with Dl was used in lane 14 for alignment of lanes 14 -18 with the other lanes. Lanes 1-13 can be aligned using minor N bands. Lanes 14 -16 and lanes 17 and 18 are from the same gel exposed to autoradiographic film for different periods due to different levels of N expression. The molecules marked by an asterisk are variably produced in these experiments. The blots containing lanes 11-18 were reprobed with an antiheat shock protein 70 antibody (Sigma) to show that the same amount of total proteins is present in these lanes. b, clone-8 cells show similar responses to treatments with Dl and Wg. c, N120 molecules accumulate in the cytoplasmic fraction whereas N350 molecules accumulate in the membrane fraction. Cells in b and c were treated for 2 h. Cells: N, N ⌬EGF1-18 and N ⌬EGF19 -36 ϭ S2 cells expressing these molecules; S2 ϭ untransfected S2 cells; Dl ϭ S2-Dl cells; Wg: Ϫ ϭ media conditioned by growth of S2 cells; ϩ ϭ media conditioned by growth of S2-Wg cells.

Delta, Wingless, and Different Notch Molecules in Cells 9100
in our experiments and the ϳ120-kDa in vivo molecule described by others are likely the same molecules. These molecules themselves act as activators of genes responsive to Dl. In numerous experiments, treatment of S2-N or clone-8 cells with Wg never resulted in accumulation of N120. Thus, it appears that N120 is not the activator of genes responsive to Wg.
The co-linear N350 molecules accumulate to higher levels in Wg-treated cells when compared with both untreated and Dltreated cells. Co-linear N molecules are proposed to be cut into separate extracellular and intracellular fragments that are noncovalently linked to produce the heterodimeric cell surface receptor (24 -26). N350 accumulates in Wg-treated cells possibly because they are converted into the heterodimeric cell surface receptors at a slower rate compared with the rate in untreated S2-N cells and Dl-treated S2-N cells. This slower rate may be due to non-processing of the intracellular domain of Wg-bound N receptors and availability of limited receptor sites in the plasma membrane. Because the N intracellular domain is barely detectable in the cytoplasmic fraction of Wgtreated cells and most remain associated with the membranes (Fig. 1c), genes responsive to Wg are likely to be activated by molecules that interact with the N intracellular domain rather than the N intracellular domain itself. N55 is unlikely to be involved in activation of Dl-or Wg-responsive genes because it is produced by both ligands. N55 is produced in response to signaling by the full-length N and is proposed to be the intracellular domain of the truncated N receptor found enriched in tissues developing after signaling by the full-length N. 3 N is a cell surface receptor, the activities of which are regulated by ligands binding its extracellular domain. Dl and Wg bind two different regions: Dl binds EGF-like repeats 11 and 12 (21), whereas Wg binds more than one site in the EGF-like repeats 19 -36 region (7). We propose that the N receptor is a "switch" for activation of different signaling pathways during development (Fig. 2). Dl binds the EGF-like repeats 11-12 region to shunt the N120⅐Su(H) complex into the nucleus for turning on the expression of Dl-related genes. Wg binds the EGF-like repeats 19 -36 region to send a transcriptional activator to the nucleus for turning on the expression of Wg-related genes.
Our results also suggest that the set of molecules involved in transducing N intracellular signals in response to Dl is likely to be different from the set that transduces N intracellular signals in response to Wg. We have identified what may be the initial differences between these two different N intracellular signaling pathways, the ones likely to set in motion different intracellular events. Starting with these differences, it should be possible in the future to identify the molecules that are involved in each N intracellular signaling pathway. This will enable integration and a better understanding of the functions of N, Dl, and Wg during Drosophila development.