title: DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.
abstract: Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: James T
authors.initials: JT
authors.lastname: Nichols
authors.forename: Alison
authors.initials: A
authors.lastname: Miyamoto
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Olsen
authors.forename: Brendan
authors.initials: B
authors.lastname: D'Souza
authors.forename: Christine
authors.initials: C
authors.lastname: Yao
authors.forename: Gerry
authors.initials: G
authors.lastname: Weinmaster
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D019281:Dimerization; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051881:Receptor, Notch1 / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D008024:Ligands; D046911:Macromolecular Substances; C497762:Notch1 protein, mouse; D051881:Receptor, Notch1; D010447:Peptide Hydrolases; D051722:ADAM Proteins