The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro

Abstract

DLL1 and DLL4 are Notch ligands with high structural similarity but context-dependent functional differences. Here, we analyze their functional divergence using cellular co-culture assays, biochemical studies, and in vivo experiments. DLL1 and DLL4 activate NOTCH1 and NOTCH2 differently in cell-based assays and this discriminating potential lies in the region between the N-terminus and EGF repeat three. Mice expressing chimeric ligands indicate that the ectodomains dictate ligand function during somitogenesis, and that during myogenesis even regions C-terminal to EGF3 are interchangeable. Substitution of NOTCH1-interface residues in the MNNL and DSL domains of DLL1 with the corresponding amino acids of DLL4, however, does not disrupt DLL1 function in vivo. Collectively, our data show that DLL4 preferentially activates NOTCH1 over NOTCH2, whereas DLL1 is equally effective in activating NOTCH1 and NOTCH2, establishing that the ectodomains dictate selective ligand function in vivo, and that features outside the known binding interface contribute to their differences.

Canonical Notch ligands and Fringes have distinct effects on NOTCH1 and NOTCH2

“…NOTCH2 had a higher affinity for DLL1 than NOTCH1, while NOTCH1 had both a higher affinity and binding maximum for DLL4 than NOTCH2. These results are in line with the relative affinities of NOTCH1 and NOTCH2 for DLL1 and DLL4 reported previously (47,48). In addition, NOTCH2 had higher affinity for JAG1 than NOTCH1, while NOTCH1 and NOTCH2 showed similar affinity for JAG2 ( Figure 1C).…”

Mechanisms of Notch signaling: a simple logic deployed in time and space

“…Whether the ICDs of Dll1 and Dll4 interact with different molecules that contribute to these distinct capacities is a possibility that remains to be explored. We note, however, that although Dll1 function cannot be replaced by Dll4 in mesodermal tissues of the mouse embryo, the ICD of Dll1 can be functionally substituted by the ICD of Dll4 in the same tissues (Tveriakhina et al, 2018). This suggests that a possible alteration in Notch dynamics by the Dll4 ICD (which causes a sustained NICD production) might be buffered in vivo by other components of the Notch response.…”

Distinctive properties of the interactions between Notch and Notch ligands

“…Using gene‐modified mice expressing Dll4 instead of Dll1 from the endogenous Dll1 locus, it was revealed that Dll4 cannot rescue the defect of Dll1 deficiency in somitogenesis, while both acting redundantly for the maintenance of the retina progenitors (Preuße et al, ; Tveriakhina et al, ). This indicated that Dll1 and Dll4 have redundant or different functions depending on the developmental context.…”

“…Their simple interaction, observed by use of their soluble forms, seems to be similar to that between Dll1 and Notch2. Recently, it was found that Dll4 and Dll1 recognizes Notch1 and Notch2 differently (Tveriakhina et al, ), however, the molecular details of this remain unclear. As such, Dll1 may possess some advantage(s) over Dll4 in the interaction with Notch2.…”