Functional Comparison between Endogenous and Synthetic Notch Systems

The Notch pathway converts receptor–ligand interactions
at the cell surface into a transcriptional response in the receiver
cell. In recent years, synthetic Notch systems (synNotch) that respond
to different inputs and transduce different transcriptional responses
have been engineered. One class of synNotch systems uses antibody–antigen
interactions at the cell surface to induce the proteolytic cleavage
cascade of the endogenous Notch autoregulatory core and the consequent
release of a synNotch intracellular domain (ICD), converting surface
antigen detection into a cellular response. While the activation of
endogenous Notch requires ubiquitylation and subsequent endocytosis
of the ligand ICD, these synNotch systems do not seem to have such
a requirement because the synNotch ligands completely lack an ICD.
This observation raises questions about existing models for the synNotch
activation mechanism. Here, we test how different structural and biochemical
factors affect the dependence of endogenous and synthetic Notch activation
on ligand ICD. We compare the behavior of antibody–antigen
synNotch (aa-synNotch) to that of endogenous Notch, and to a synNotch
system that uses rapamycin induced dimerization of FK506 binding protein
(FKBP) and FKBP rapamycin binding (FRB) domaindimerization domains
(ff-synNotch), which still requires a ligand ICD. We found that differences
in receptor–ligand affinity, in the identity of the transmembrane
domain, or in the presence or absence of extracellular epidermal growth
factor repeats cannot explain the differences in ligand ICD requirement
that distinguishes aa-synNotch from endogenous Notch or ff-synNotch.
We also found that unlike endogenous Notch and ff-synNotch, the aa-synNotch
system does not exhibit trans-endocytosis of the receptor extracellular
domain into the sender cell. These findings suggest that the aa-synNotch
systems bypass the ligand ICD requirement because antigen–antibody
pairs are able to promote other adhesive cell–cell interactions
that provide the mechanical tension needed for ligand activation.