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: Using the shedding-resistant 6xD mutant, we next addressed whether there are any correlations between the shedding of DDR1 ectodomain and the regulation of DDR1 phosphorylation. We established HEK293 cell lines stably expressing DDR1-WT or 6xD mutant. Both WT and 6xD were phosphorylated at 1 h after collagen stimulation (Figure 8, A, WT and PY, and B). We used a lower concentration of collagen (20 μg/ml) in this experiment, as adding 100 μg/ml of collagen resulted in formation of a thin collagen layer over the cells, causing cells to attach to the collagen layer rather than the bottom of plastic wells (unpublished data). Because this experiment involved washing out excess collagen, this was not ideal. We found that 20 μg/ml collagen was enough to cause DDR1 phosphorylation, and washing the cells did not detach them from the dish. The level of collagen-induced phosphorylation of DDR1-WT peaked at 1 h and then decreased upon washing out of collagen from the medium, with a half-life of ∼1.5 h (Figure 8, A, WT and PY, and B). The level of DDR1-WT in cell lysates also decreased over the incubation time (Figure 8A, WT, Cell), which inversely correlated with the accumulation of shed DDR1 in the medium (Figure 8, A, WT and Med, and C). In contrast, phosphorylation of DDR1-6xD was sustained much longer than WT, with a half-life of ∼5 h (Figure 8, A, 6xD and PY, and B). This was accompanied by noticeably lower shedding of the DDR1 ectodomain (Figure 8, A, 6xD and Med, and C). These data suggest that ectodomain shedding of DDR1 is an important process to down-regulate its phosphorylation state of the receptor.
Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.