Members of the Notch/LIN-12/GLP-1 family of transmembrane receptors are believed to play a central role in development by regulating cell-fate decisions in invertebrates. The applicant has isolated several genes encoding Notch receptors and Notch ligands from rat, and has been studying their expression and function to address the molecular mechanism of Notch signaling during mammalian development. Notch is associated with malignancies, suggesting a role for maintaining an undetermined state during normal development, and Notch is also involved in human disorders of stroke and dementia (CADASIL and Alzheimer's disease), suggesting a role outside of development as well. Experiments proposed are based on the applicant's previous findings, and are logical extensions designed to answer a number of basic questions concerning Notch function. They will determine if Notch functions as a dimer or monomer, and if ligand activation of Notch involves ligand dimerization. They also will explore how Notch cell surface expression is regulated and investigate the hypothesis that Notch is cleaved and transported to the nucleus following ligand binding. In contrast to what has been reported for the invertebrate systems, they made the surprising observation that the mammalian ligands are not equal in their ability to activate the different Notch receptors. Based on these observations, they have designed experiments to identify sequences present in the ligands and receptors that account for the observed ligand specificity. In addition, they will explore the production and use of soluble forms of Notch ligands to study Notch signaling. They found that Notch signaling leads to activation of both CBF1-dependent and CBF1-independent pathways. Preliminary data indicate that the CBF1-dependent pathway positively regulates the expression of Notch. They will investigate the molecular mechanisms underlying this positive feedback system. They will also examine the possibility of reciprocal signaling in the ligand-expressing cell following ligand-receptor interactions. Together the experiments proposed in this grant will provide new information on the structure/function requirements of Notch-ligand interactions and thereby extend our present understanding of mammalian Notch signaling.
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