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: We postulated that a potential reason for weak sensitivity of ADAM10-dependent DDR1 shedding to TIMP-3 inhibition may be due to steric hindrance caused by complex formation between ADAM10 and DDR1. To examine this possibility, we cotransfected HEK293 cells with DDR1-NF and nontagged ADAM10 and subjected the cell lysates to coimmunoprecipitation using anti-FLAG beads. As shown in Figure 4A (FLAG-immunoprecipitation, bottom), ADAM10 was coimmunoprecipitated with DDR1-NF. A similar result was obtained by coimmunoprecipitation of DDR1 with FLAG-tagged full-length ADAM10 (ADAM10-F) or ADAM10-F without metalloproteinase domain (ADAM10∆MP-F; Figure 4B), suggesting that ADAM10 is indeed in a complex with DDR1, and the catalytic metalloproteinase domain of ADAM10 is not essential for this interaction. We next investigated whether endogenous ADAM10 can form a complex with endogenous DDR1 in A431 cells. In these experiments, A431 cells were surface biotinylated before lysis of the cells and subjected to immunoprecipitation using anti-DDR1 antibody or anti-ADAM10 antibody. This two-step affinity precipitation, which involves initial precipitation with anti-DDR1 or anti-ADAM10 followed by streptavidin beads, allowed us to detect cell surface complexes of DDR1 and ADAM10. As shown in Figure 4C (Input, bottom), both proADAM10 and active ADAM10 were detected in A431 cells. The data indicate that both proform and active form of ADAM10 were coimmunoprecipitated with endogenous DDR1, suggesting that endogenous ADAM10 and DDR1 form a complex on the cell surface.
Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.