ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1).
Bottom Line: DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner.DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1.Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor.
Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.Show MeSH
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Mentions: There are two potential mechanisms to explain collagen-induced shedding of DDR1. First, collagen-induced DDR1 signaling may trigger the functional activation of ADAM10 (Vogel, 2002); second, the orientation of DDR1 may be altered upon collagen binding, which allows ADAM10 to cleave DDR1. We first investigated whether collagen-induced DDR1 signaling plays a role. We used a kinase-dead mutant of DDR1 (DDR1-KD) and a cytoplasmic domain deletion mutant (DDR1∆C), both of which are not able to transmit collagen signals (Figure 6A). We confirmed that those mutants are expressed on the cell surface in a similar manner to wild-type (WT) DDR1 by a surface biotinylation assay (Supplemental Figure S4A). As shown in Figure 6B, upon collagen stimulation, the ectodomain of DDR1-KD and DDR1∆C was shed in an efficient manner. We also examined the effect of a tyrosine kinase inhibitor, dasatinib, which inhibits DDR1 with an IC50 of 1.35 nM (Day et al., 2008). As shown in Figure 6C, dasatinib treatment at 50 nM did not influence DDR1 shedding, whereas it completely inhibited phosphorylation of DDR1 upon collagen stimulation. These data strongly suggest that collagen-induced DDR1 shedding is not dependent on DDR1 signaling.
Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.