Mouse Jagged1 physically interacts with Notch2 and other Notch receptors -Assessment by quantitative methods

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors, However, the physical interaction between D SL proteins and Notch receptors is poorly understood, In this study, we clo ned a cDNA for mouse Jagged1 (mJagged1), To identify the receptor interacti ng with mJagged1 and to gain insight into its binding characteristics, we e stablished two experimental systems using fusion proteins comprising variou s extracellular parts of mJagged1, a "cell" binding assay and a "solid-phas e" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on th e cell surface and to a purified extracellular portion of mNotch2, respecti vely, in a Ca2+-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation con stants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-bin ding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is in dispensable for binding to mNotch2, The epidermal growth factor-like repeat s of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role, Finally, solid-phase binding assay sho wed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesti ng that mJagged1 is a ligand for multiple Notch receptors.