title: Glycosylation at Asn211 regulates the activation state of the discoidin domain receptor 1 (DDR1).
abstract: Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn(211) residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved (211)NDS N-glycosylation motif, but not other N-glycosylation sites (Asn(260), Asn(371), and Asn(394)), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr(484), Tyr(520), Tyr(792), and Tyr(797). The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved (211)NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.
authors: From the Department of Pathology, Wayne State University, Detroit, Michigan 48201.
authors: Hsueh-Liang
authors: HL
authors: Fu
authors: Rajeshwari R
authors: RR
authors: Valiathan
authors: Leo
authors: L
authors: Payne
authors: Malika
authors: M
authors: Kumarasiri
authors: Kiran V
authors: KV
authors: Mahasenan
authors: Shahriar
authors: S
authors: Mobashery
authors: Paul
authors: P
authors: Huang
authors: Rafael
authors: R
authors: Fridman
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D002460:Cell Line; D024042:Collagen Type I / Q000494:pharmacology; D017124:Conserved Sequence; D000070819:Discoidin Domain Receptor 1; D004705:Endocytosis / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D024042:Collagen Type I; D001216:Asparagine; C092030:DDR1 protein, human; D000070819:Discoidin Domain Receptor 1; D020794:Receptor Protein-Tyrosine Kinases
keywords: Collagen; Glycosylation; Mutagenesis Site-specific; Receptor Regulation; Receptor Tyrosine Kinase