Developing a mouse model to study Notch furin processing

University Of California Los Angeles, Los Angeles CA

Investigators

Abstract

DESCRIPTION (provided by applicant): The Notch cell surface receptor is activated by ligands expressed on the surface of neighboring cells to direct correct cell specification, patterning, and morphogenesis within virtually all metazoan organisms studied to date. Therefore it is not surprising that Notch signaling has been linked to certain cancers, inherited human syndromes and neurodegenerative diseases. A model for Notch signaling has been proposed in which ligand binding induces proteolysis of Notch to liberate its intracellular domain, which then functions as a signal transducer through direct interaction and activation of the transcriptional regulator CSL. Induction of CSL-dependent signaling occurs following ligand binding to a heterodimeric form of Notch that is generated through proteolytic processing by furin. In addition to CSL-dependent Notch signaling we have identified another pathway that functions independently of CSL activation. Biotinylation studies have indicated that in addition to heterodimeric Notch1, uncleaved full-length Notch1 exists at the cell surface, suggesting that this isoform might also function in ligand binding and activation of signal transduction. Consistent with this idea, functional analysis in cell culture indicates that uncleaved full-length cell surface Notch1 blocks myogenic differentiation in response to ligand, in the absence of CSL activation. To study CSL-independent Notch signaling in intact animals this application outlines experiments designed to generate "knock-in" (Ki) mice in which the Notch1 furin-cleavage site has been deleted so that only uncleaved full-length Notch1 is expressed on the surface of homozygous mutant cells. Biochemical and phenotypic characterization of these Notch1 mutant animals will not only document the biological relevance of CSL-independent Notch signaling and confirm that uncleaved full-length Notch serves as a receptor for this pathway, but it should also allow the identification of cell types and processes regulated by this pathway.