The Notch receptor mediates cell fate decisions in organisms as diverse as flies and humans. The observation that mutations in Notch are associated with neoplasias and Alagille and CADASIL syndromes, underscore its significance. Transmembrane proteins, Delta and Serrate, function as activating ligands for the Notch receptor in Drosophila. The recent report of Delta cleavage, by Kuzbanian, an ADAM family metalloprotease, to release an extracellular fragment (D1/EC) adds a new dimension to Notch signaling as the membrane-anchored and soluble forms may elicit different responses. The proposed study aims to discern between an agonistic or antagonistic role for soluble Delta in Notch signaling, in vitro and in vivo. The working hypothesis proposed is that D1/EC can activate Notch. Further, Kuzbanian cannot cleave Serrate and this specificity arises due to the effect of Fringe (a galactosyltransferase) on Serrate, thus conferring ligand specificity during Notch signaling. This hypothesis will be tested by: 1) Identification of the Kuzbanian cleavage site on Delta. Determination of whether Kuzbanian can cleave Serrate; 2) Analyzing the functional role of Delta cleavage in vitro and in vivo; and 3) Analyzing the role of Fringe in the cleavage of Delta and Serrate. Delta cleavage site will be identified by PCR-based site-directed mutagenesis and domain- swap experiments. The role of Delta cleavage will be assessed in vitro and in a Cell culture based transcription assay; and in vivo by testing the ability of an uncleavable form of Delta to rescue Delta phenotypes in Drosophila. The ability of D1/EC to activate the Notch pathway will be tested by expressing D1/EC in the eye and the wing disc using the UAS/GAL4 system and assessing its ability to induce gain-of-function phenotypes in Drosophila. The role of Fringe in Serrate cleavage will be assessed in a Cell culture based system.
