title: Intrinsic selectivity of Notch 1 for Delta-like 4 over Delta-like 1.
abstract: Notch signaling makes critical contributions to cell fate determination in all metazoan organisms, yet remarkably little is known about the binding affinity of the four mammalian Notch receptors for their three Delta-like and two Jagged family ligands. Here, we utilized signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Systematic deletion mutagenesis of the human Notch1 ectodomain revealed that epidermal growth factor (EGF) repeats 6-15 are sufficient to maintain signaling in a reporter assay at levels comparable with the full-length receptor, and identified important contributions from EGF repeats 8-10 in conveying an activating signal in response to either Dll1 or Dll4. Truncation studies of the Dll1 and Dll4 ectodomains showed that the MNNL-EGF3 region was both necessary and sufficient for full activation. Plate-based and cell binding assays revealed a specific, calcium-dependent interaction between cell-surface and recombinant Notch receptors and ligand molecules. Finally, direct measurement of the binding affinity of Notch1 EGF repeats 6-15 for Dll1 and Dll4 revealed that Dll4 binds with at least an order of magnitude higher affinity than Dll1. Together, these studies give new insights into the features of ligand recognition by Notch1, and highlight how intrinsic differences in the biochemical behavior of receptor-ligand complexes can influence receptor-mediated responses of developmental signaling pathways.
authors.affiliation: From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,; the Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02115, and.
authors.forename: Marie Blanke
authors.initials: MB
authors.lastname: Andrawes
authors.affiliation: From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,; the Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02115, and.
authors.forename: Xiang
authors.initials: X
authors.lastname: Xu
authors.affiliation: the Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02115, and.
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.affiliation: From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,; the Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02115, and.
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.affiliation: From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,; the Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02115, and.
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
authors.affiliation: the Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02114.
authors.forename: Jon C
authors.initials: JC
authors.lastname: Aster
authors.affiliation: From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,; the Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02115, and. Electronic address: Stephen_blacklow@hms.harvard.edu.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Blacklow
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C512941:DLK1 protein, human; C424126:DLL4 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1
keywords: Biolayer Interferometry; Cell Signaling; Cell Surface Receptor; Cell-Cell Interaction; Notch Pathway; Receptor Structure-Function; Receptor-Ligand Interaction; Recombinant Protein Expression