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ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1).

Shitomi Y, Thøgersen IB, Ito N, Leitinger B, Enghild JJ, Itoh Y - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.

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Coimmunoprecipitation of DDR1 with ADAM10. (A) HEK293 cells were cotransfected with DDR1-NF, wild-type ADAM10, or ADAM10ΔMP in combination as indicated and subjected to immunoprecipitation with anti-FLAG affinity beads. Bound materials were analyzed by Western blotting using anti–DDR1 ectodomain or anti-ADAM10 antibodies. Asterisks indicate the protein that was pulled down. MP, metalloproteinase domain. (B) HEK293 cells were transiently cotransfected with FLAG-tagged ADAM10 (ADAM10-F), ADAM10ΔMP-F, or DDR1 in combination as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by Western blotting with anti-DDR1 ectodomain or anti-ADAM10 antibodies. DDR1 was coimmunoprecipitated with ADAM10-F or ADAM10ΔMP-F (FLAG-immunoprecipitation, top). Asterisks indicate the protein that was pulled down. (C) Coimmunoprecipitation of endogenous DDR1 and ADAM10. A431 cells were subjected to cell surface biotinylation before cell lysis. Cell lysates were subjected to two-step affinity precipitation using antixDDR1 ectodomain or anti-ADAM10 ectodomain conjugated to protein G–coated Dynabeads followed by streptavidin beads. Coimmunoprecipitated DDR1 and ADAM10 bound to streptavidin beads were visualized by Western blotting using anti-DDR1 C-terminus or anti-ADAM10 antibodies. Control sample was incubated with protein G–Dynabeads without antibodies. Active, active form of ADAM10; Pro, proform. Blank lanes that were cropped out of the blot are indicated by black lines.
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Figure 4: Coimmunoprecipitation of DDR1 with ADAM10. (A) HEK293 cells were cotransfected with DDR1-NF, wild-type ADAM10, or ADAM10ΔMP in combination as indicated and subjected to immunoprecipitation with anti-FLAG affinity beads. Bound materials were analyzed by Western blotting using anti–DDR1 ectodomain or anti-ADAM10 antibodies. Asterisks indicate the protein that was pulled down. MP, metalloproteinase domain. (B) HEK293 cells were transiently cotransfected with FLAG-tagged ADAM10 (ADAM10-F), ADAM10ΔMP-F, or DDR1 in combination as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by Western blotting with anti-DDR1 ectodomain or anti-ADAM10 antibodies. DDR1 was coimmunoprecipitated with ADAM10-F or ADAM10ΔMP-F (FLAG-immunoprecipitation, top). Asterisks indicate the protein that was pulled down. (C) Coimmunoprecipitation of endogenous DDR1 and ADAM10. A431 cells were subjected to cell surface biotinylation before cell lysis. Cell lysates were subjected to two-step affinity precipitation using antixDDR1 ectodomain or anti-ADAM10 ectodomain conjugated to protein G–coated Dynabeads followed by streptavidin beads. Coimmunoprecipitated DDR1 and ADAM10 bound to streptavidin beads were visualized by Western blotting using anti-DDR1 C-terminus or anti-ADAM10 antibodies. Control sample was incubated with protein G–Dynabeads without antibodies. Active, active form of ADAM10; Pro, proform. Blank lanes that were cropped out of the blot are indicated by black lines.

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.


ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1).

Shitomi Y, Thøgersen IB, Ito N, Leitinger B, Enghild JJ, Itoh Y - Mol. Biol. Cell (2014)

Coimmunoprecipitation of DDR1 with ADAM10. (A) HEK293 cells were cotransfected with DDR1-NF, wild-type ADAM10, or ADAM10ΔMP in combination as indicated and subjected to immunoprecipitation with anti-FLAG affinity beads. Bound materials were analyzed by Western blotting using anti–DDR1 ectodomain or anti-ADAM10 antibodies. Asterisks indicate the protein that was pulled down. MP, metalloproteinase domain. (B) HEK293 cells were transiently cotransfected with FLAG-tagged ADAM10 (ADAM10-F), ADAM10ΔMP-F, or DDR1 in combination as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by Western blotting with anti-DDR1 ectodomain or anti-ADAM10 antibodies. DDR1 was coimmunoprecipitated with ADAM10-F or ADAM10ΔMP-F (FLAG-immunoprecipitation, top). Asterisks indicate the protein that was pulled down. (C) Coimmunoprecipitation of endogenous DDR1 and ADAM10. A431 cells were subjected to cell surface biotinylation before cell lysis. Cell lysates were subjected to two-step affinity precipitation using antixDDR1 ectodomain or anti-ADAM10 ectodomain conjugated to protein G–coated Dynabeads followed by streptavidin beads. Coimmunoprecipitated DDR1 and ADAM10 bound to streptavidin beads were visualized by Western blotting using anti-DDR1 C-terminus or anti-ADAM10 antibodies. Control sample was incubated with protein G–Dynabeads without antibodies. Active, active form of ADAM10; Pro, proform. Blank lanes that were cropped out of the blot are indicated by black lines.
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Related In: Results  -  Collection

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Figure 4: Coimmunoprecipitation of DDR1 with ADAM10. (A) HEK293 cells were cotransfected with DDR1-NF, wild-type ADAM10, or ADAM10ΔMP in combination as indicated and subjected to immunoprecipitation with anti-FLAG affinity beads. Bound materials were analyzed by Western blotting using anti–DDR1 ectodomain or anti-ADAM10 antibodies. Asterisks indicate the protein that was pulled down. MP, metalloproteinase domain. (B) HEK293 cells were transiently cotransfected with FLAG-tagged ADAM10 (ADAM10-F), ADAM10ΔMP-F, or DDR1 in combination as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by Western blotting with anti-DDR1 ectodomain or anti-ADAM10 antibodies. DDR1 was coimmunoprecipitated with ADAM10-F or ADAM10ΔMP-F (FLAG-immunoprecipitation, top). Asterisks indicate the protein that was pulled down. (C) Coimmunoprecipitation of endogenous DDR1 and ADAM10. A431 cells were subjected to cell surface biotinylation before cell lysis. Cell lysates were subjected to two-step affinity precipitation using antixDDR1 ectodomain or anti-ADAM10 ectodomain conjugated to protein G–coated Dynabeads followed by streptavidin beads. Coimmunoprecipitated DDR1 and ADAM10 bound to streptavidin beads were visualized by Western blotting using anti-DDR1 C-terminus or anti-ADAM10 antibodies. Control sample was incubated with protein G–Dynabeads without antibodies. Active, active form of ADAM10; Pro, proform. Blank lanes that were cropped out of the blot are indicated by black lines.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, United Kingdom.

Show MeSH
Related in: MedlinePlus