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

A small number of signaling systems control how an animal develops from a single cell into a complex organism made up of many different cell types. Signals pass back and forth between cells, switching genes on and off to direct the development of tissues and organs. One of these signaling systems, called Notch, is so ancient that it appears in nearly all multicellular organisms. A cell sends a Notch signal using proteins called Delta or Jagged ligands that span membrane of the cell, so that part of the protein sits inside the cell and part remains outside. To change the behavior of another cell, the ligands bind to proteins called Notch receptors that span the membrane of the receiving cell. Mammals have two types of Delta ligand, two types of Jagged ligand and four types of Notch receptor. Cells in different tissues display different combinations of these eight proteins. Two Delta ligands called DLL1 and DLL4 often appear together in developing organisms. Some tissues need both and some only the one or the other. In some cases one ligand can compensate if the other is missing, but in others not. It was not clear why this is, or which parts of the proteins are responsible. Tveriakhina et al. used mouse cells to investigate how DLL1 and DLL4 interact with two Notch receptors, called NOTCH1 and NOTCH2. The results of these experiments show that while DLL1 can bind and activate both Notch receptors equally, DLL4 prefers to partner with NOTCH1. To find out which parts of the ligands are responsible for this selectivity, Tveriakhina et al. created hybrid ligands that contained a mixture of regions from DLL1 and DLL4. These suggest that the different binding preferences depend on parts of the ligands that sit outside cells and that lie outside the known sites of binding contact with the Notch receptors. Further experiments studied mice that had been engineered to produce hybrid ligands as replacements for DLL1. A hybrid ligand consisting of the part of DLL1 that sits outside cells and the part of DLL4 found inside cells generated Notch signals in the tissue that depended on the activity of DLL1. However, a hybrid consisting of the part of DLL4 that sits outside cells and the part of DLL1 found inside cells did not, showing that in developing mice the parts that sit outside the cells contribute to the different functions of DLL1 and DLL4. Overall, the results presented by Tveriakhina et al. show that interactions between specific ligands and receptors play important roles in how mammals develop. Further efforts to understand which parts of the ligands affect selectivity could ultimately allow researchers to develop ways to modify how ligands and receptors interact. Such “molecular engineering” strategies could enable cell responses to be precisely controlled by pairing designer ligand-receptor pairs to develop cell-based therapies.