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 investigate collagen-induced ectodomain shedding of DDR1, we transiently transfected human embryonic kidney 293 (HEK293) cells with an expression plasmid for N-terminally FLAG-tagged DDR1 (DDR1-NF) or mock vector, and stimulated them with collagen I. After 24 h, a significant amount of the shed form of DDR1-NF with a molecular size of 60 kDa was detected in the conditioned medium of collagen-treated cells, which was accompanied by reduced levels of 120-kDa, full-length DDR1 and increased levels of the receptor remnant, the so-called C-terminal fragment (CTF), in comparison to untreated cells (Figure 1A). Collagen-induced DDR1 shedding was also confirmed in cells expressing endogenous DDR1, including A431 squamous cell carcinoma and MCF-7 human breast carcinoma cell lines (Figure 1B). The molecular size of full-length and shed DDR1 in MCF-7 was slightly lower than that in A431, which might be the result of different posttranslational modifications such as glycosylation or expression of different DDR1 isoforms. We observed two shed fragments of DDR1 in the medium of MCF-7 upon collagen stimulation. The reason for the appearance of the two forms of shed DDR1 in MCF-7 cells is not clear. We also detected minor spontaneous DDR1 shedding without collagen stimulation (Figure 1, –Collagen), but collagen-induced DDR1 shedding occurred at significantly higher levels. The appearance of the CTF is inconsistent between the cell lines, which may be attributed to different levels of clearance of the CTF during the 24-h period of culture. Taken together, these results suggest that collagen-induced DDR1 shedding is likely to be a common property of DDR1-expressing cells.
Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.