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: To search for a potential proteinase involved in DDR1 ectodomain shedding, we examined the effect of inhibitors for different classes of proteinases, including metalloproteinase inhibitors GM6001 and Marimastat (Mst), a cysteine proteinase inhibitor (E-64), an aspartic proteinase inhibitor (pepstatin A), a serine proteinase inhibitor (AEBSF), and a γ-secretase inhibitor X (L-685, 458). Among them, only the broad-spectrum metalloproteinase inhibitors GM6001 and Mst inhibited shedding of DDR1 (Figure 2A), confirming that a metalloproteinase is responsible, as previously reported (Vogel, 2002). To characterize further metalloproteinase activity, we examined the effect of tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, and TIMP-3 on DDR1 shedding. We found that addition of TIMP-1 or TIMP-2 at 500 nM did not influence the shedding in HEK293 cells and A431 cells (Supplemental Figure S1, A and B). It was reported that overexpression of TIMP-3 inhibited DDR1 shedding by 75% in HEK293 cells (Slack et al., 2006). Our data also showed that addition of purified TIMP-3 at 100, 200, and 300 nM partially inhibited DDR1 shedding in HEK293 cells by 21, 30, and 41%, respectively (Figure 2B). We also examined effects of both stable and transient expression of TIMP-3 in HEK293. However, TIMP-3 overexpression did not inhibit DDR1 shedding in our experimental conditions (Supplemental Figure S1, B and C). We speculate that this lack of inhibition may be due to insufficient levels of TIMP-3 produced from those transfected cells. These results suggest that DDR1 shedding can be inhibited by TIMP-3, but it requires high concentration for inhibition of DDR1 shedding in the HEK293 cell system. To examine whether this is also the case for endogenous DDR1 shedding, we analyzed the effect of TIMP-3 in A431 cells. In contrast to HEK293 cells, endogenous DDR1 shedding was not inhibited by TIMP-3 even at 300 nM (Figure 2C). We were unable to examine a higher concentration of TIMP-3 due to the difficulty of TIMP-3 purification. The inability of TIMP-3 to inhibit DDR1 shedding was unlikely due to loss of bioavailability of TIMP-3, which can be caused by its endocytosis through LRP1 (Scilabra et al., 2013), as significant amounts of TIMP-3 (∼80% remained for each concentration of TIMP-3) were detected in the medium even after 24 h of incubation (Figure 2C). We thus concluded that collagen-induced endogenous DDR1 shedding is insensitive to TIMPs, including TIMP-3 in A431 cells.
Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.