Characterization and comparison of protein complexes initiated by the intracellular domain of individual Notch paralogs.

The Notch signaling pathway is essential for embryonic development, organogenesis, and tissue homeostasis. Aberrant Notch signaling is associated with several types of cancers. The active form of Notch receptor is its intracellular domain (NICD), which is released from the cell membrane by serial proteolytic cleavages following ligand binding. Dose-dependent effects of NICD on cellular phenotypes have been observed under several conditions although the underlying mechanisms have not been well studied. Moreover, there are four mammalian Notch paralogs that have redundant as well as unique functions. The molecular basis for this variability is also not well understood. In this study, we used size exclusion chromatography to examine the overall distribution of NICD among NICD-containing protein complexes under conditions of increasing NICD abundance. We found that the assembly of NICD protein complexes was dose-dependent and that the abundance of the canonical complex was limited by, MAML, one of the proteins involved in the formation of canonical NICD transactivation complex, which became saturated with increasing NICD abundance. In addition, N4ICD showed a unique elution profile among the four NICDs. These results help to explain the dose-dependent and paralog-specific activities of NICD. These results are informative for the development of new reagents to block Notch signaling for therapeutic benefit.