Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation.
The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs [EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is ~10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Ankur
A
Sharma
Annapoorni
A
Rangarajan
Rajan R
RR
Dighe
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D001667:Binding, Competitive / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D051881:Receptor, Notch1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D008024:Ligands; D010455:Peptides; D051881:Receptor, Notch1; D004815:Epidermal Growth Factor; D002118:Calcium