Structural and Biochemical Mechanisms of Notch Signal Activation and Inhibition

Abstract

Cell signaling encompasses the fundamental processes which enable an organism to perceive and respond to its environment. Productive cell signaling occurs in a series of tightly regulated steps that require stimulus detection, signal transmission, and a downstream response. The Notch signaling pathway is an essential cell-cell communication system in metazoans that has critical roles in cell fate determination, growth and development. In mammals, there are two families of Notch-activating ligands, Jagged and Delta-like. The Delta-like paralogues, DLL1 and DLL4, have high structural similarity but exhibit context-dependent functional differences. Notch pathway activation induces transcription of Notch-responsive genes including the Notch regulated ankyrin repeat protein (NRARP) which acts as a feedback inhibitor of the Notch response and is among the most commonly Notch-induced genes in many cell types. In this thesis, I describe the molecular basis for functional divergence of the highly similar ligands DLL1 and DLL4 and a molecular mechanism for NRARP-mediated feedback inhibition of Notch signaling. First, I analyze the functional divergence of DLL1 and DLL4 using cellular assays and biochemical studies. 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. Our results reveal that DLL4 preferentially activates NOTCH1 over NOTCH2, whereas DLL1 exhibits no preference for either receptor. These studies establish that ligand ectodomains dictate selective function and features outside the receptor-binding interface contribute to functional differences. Next, I show that NRARP exerts its inhibitory effect by binding directly to the Notch transcriptional activation complex (NTC), requiring both the RBPJ transcription factor and Notch intracellular domain, but not Mastermind-like proteins or DNA. The X-ray structure of a NRARP/RBPJ/NOTCH1/DNA complex reveals that assembly of NRARP/RBPJ/NOTCH1 complexes relies on simultaneous engagement of RBPJ and NOTCH1 in a non-canonical binding mode involving the extension of the NOTCH1 ankyrin repeat stack by the ankyrin repeats of NRARP. Interface mutations of NRARP disrupt entry into NTCs and abrogate feedback inhibition of Notch signaling. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.