Unravelling Paralog-Specific Notch Signaling through Ternary Complex Stability and Transcriptional Activation Measurements Using Chimeric Receptors

Abstract

Notch signaling is mediated by four paralogous receptors with conserved architectures and overlapping yet non-redundant functions. Notch signaling generates a transcriptional activation complex (NTC) wherein the N-terminal RAM and ankyrin repeat (ANK) regions of the Notch intracellular domain (NICD) bind transcription factor CSL and recruit co-activator Mastermind-like (MAML). To better understand paralog-specific differences in Notch signaling, we analyzed the thermodynamics of binary and ternary NTCs for all four Notch paralogues and chimeric constructs. We find that RAMANK:CSL stability ({Delta}GRA) is primarily determined by the RAM region while MAML binding to preformed RAMANK:CSL complexes ({Delta}GMAML) is largely determined by the ANK region. We determined transcriptional activation data for the paralogous and chimeric NICDs and analyzed the data with an independent multiplicative model. This analysis shows ternary complex stability ({Delta}GTC, where {Delta}GTC= {Delta}GRA+{Delta}GMAML) correlates well with transcriptional activations and provides insights into contributions of RAM, ANK and the C-terminal regions to Notch signaling.