Regions of Drosophila Notch That Contribute to Ligand Binding and the Modulatory Influence of Fringe

Kenneth D. Irvine,Liang Lei,Aiguo Xu

doi:10.1074/jbc.m505569200

Kenneth D. Irvine, Liang Lei + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m505569200

Copy DOI

Abstract

Two glycosyltransferases that transfer sugars to epidermal growth factor (EGF) domains, OFUT1 and Fringe, regulate Notch signaling. To characterize the impact of glycosylation at the 23 consensus O-fucose sites in Drosophila Notch, we conducted deletion mapping and site-specific mutagenesis and then assayed the binding of soluble forms of Notch to cell-surface ligands. Our results support the conclusion that EGF11 and EGF12 are essential for ligand binding, but indicate that other EGF domains also make substantial contributions to ligand binding. Characterization of Notch deletion constructs and O-fucose site mutants further revealed that no single site or region can account for the influence of Fringe on Notch-ligand binding. Additionally, we observed an influence of Fringe on a Notch fragment including only 4 of its 36 EGF domains (EGF10-13). Together, our observations imply that glycosylation influences Notch-ligand interactions through a distributive mechanism that involves local interactions with multiple EGF domains and led us to suggest a structural model for how Notch interacts with its ligands.

Highlights

Notch proteins are transmembrane receptors for an intercellular signaling pathway that affects numerous cell fate decisions throughout the Metazoa
Multiple epidermal growth factor (EGF) Domains Contribute to Notch-Ligand Binding—The observation that Fng can influence Notch-ligand binding even when the only O-fucose site in the previously
Based on the knowledge that EGF11 and EGF12 are essential for ligand binding and a concern that adjacent EGF domains might influence the stability or folding of EGF11 and EGF12, we created a polypeptide including EGF10 –13 of Notch fused to alkaline phosphatase (N:AP-EGF10 –13) (Fig. 2C)

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—Plasmids expressing truncated versions of the extracellular domain of Notch fused to alkaline phosphatase (AP; referred to as N:AP) were created using a PCR cloning strategy. Plasmid pMT-WB/N-AP was constructed by cloning a 6.3-kb EcoRIXbaI fragment including Notch EGF domains fused to AP from pRmHa3/N-AP [20] into pMT-WB. The binding assay involved mixing conditioned medium containing N:AP or Fc:AP (a control protein consisting of the Fc domain of human IgG fused to AP) with S2 cells expressing a Notch ligand or with control S2 cells. To normalize for variation in transfection efficiency and secretion of different constructs, the amount of fusion protein in the medium was assayed by measuring AP activity and was equalized among experiments presented in the same table by the addition of conditioned medium from untransfected S2 cells (to dilute) or by centrifugation though Amicon Ultra centrifugal filters (Mr 10,000 cutoff; Millipore Corp.) (to concentrate). S2 cells do not endogenously express significant levels of Serrate or Delta, and S2 cells transiently transfected with vector (pRMHa-3) were used as a negative control for ligand binding (control S2 cells). Sequence Comparisons—The conservation of O-fucose sites and Ca2ϩ-binding sites was assessed by comparing the sequences of Notch from Drosophila melanogaster (GenBankTM accession number P07207), Lucilia cuprina (accession number AAC36151), Boophilus microlus (accession number AAN06819), Lytechinus variegatus (gi 7512075), Takifugu rubripes (accession number BAA20535), Danio rerio (gi 18859115), Halocynthia roretzi (gi 7522619), Branchiostoma floridae (gi 12057020), and Xenopus laevis (gi 1709335); Notch from Mus musculus (gi 6679092), Rattus norvegicus (gi 6093542), and Homo sapiens (gi 27894368); and Notch from H. sapiens (gi 24041035), M. musculus (gi 20138876), and R. norvegicus (gi 13242247) using C2XXX(A/G/S)(S/ T)C3 as a consensus O-fucose site and (D/N/E)X(D/N/E)(E/D/Q)C1 . . . C3X(D/N)XXXX(Y/F)XC4 as a consensus Ca2ϩ-binding site, where C1, C2, C3, and C4 are the first, second, third, and fourth cysteines of an EGF domain

TABLE I Binding of Notch deletion constructs

Fc:AP

TABLE II Binding of Notch point mutations

TABLE III Binding of additional Notch point mutations

DISCUSSION

TABLE IV Binding of a minimal Notch fragment