Limits...
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.

Show MeSH

Related in: MedlinePlus

Collagen binding, but not DDR1 phosphorylation, is required for DDR1 shedding. (A) Schematic representation of mutant DDR1 constructs used in the experiments. DD, discoidin-homology domain; ∆C, cytoplasmic domain-deleted; DLD, discoidin-like domain; FLAG, FLAG tag (DYKDDDDK); HA, HA tag (YPYDVPDYA); JM, juxtamembrane region; KD, kinase dead; S, signal peptide; TM, transmembrane domain; TKD, tyrosine kinase domain. (B) HEK293 cells expressing DDR1-WT (WT), DDR1-KD (KD), or DDR1∆C (∆C) were treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1 ectodomain (Med and Cell), anti–phosphotyrosine 4G10 (PY), or anti-actin. Asterisk indicates tyrosine-phosphorylated proteins other than DDR1. (C) HEK293 cells expressing DDR1-WT were treated with collagen I in the presence or absence of 50 nM dasatinib or vehicle control (DMSO) for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), antixDDR1 C-terminus (Cell), anti-PY, or anti-actin. (D) HEK293 cells were transiently transfected as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti-DDR1 ectodomain and anti-actin antibodies. For the phosphotyrosine blot, cells treated with collagen for 1 h were used. The relative intensities of shed DDR1 are shown at the bottom of the top panel. (E) HEK293 cells were transfected with N-terminally FLAG-tagged DDR1 mutants as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were analyzed by Western blotting as in D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4325837&req=5

Figure 6: Collagen binding, but not DDR1 phosphorylation, is required for DDR1 shedding. (A) Schematic representation of mutant DDR1 constructs used in the experiments. DD, discoidin-homology domain; ∆C, cytoplasmic domain-deleted; DLD, discoidin-like domain; FLAG, FLAG tag (DYKDDDDK); HA, HA tag (YPYDVPDYA); JM, juxtamembrane region; KD, kinase dead; S, signal peptide; TM, transmembrane domain; TKD, tyrosine kinase domain. (B) HEK293 cells expressing DDR1-WT (WT), DDR1-KD (KD), or DDR1∆C (∆C) were treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1 ectodomain (Med and Cell), anti–phosphotyrosine 4G10 (PY), or anti-actin. Asterisk indicates tyrosine-phosphorylated proteins other than DDR1. (C) HEK293 cells expressing DDR1-WT were treated with collagen I in the presence or absence of 50 nM dasatinib or vehicle control (DMSO) for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), antixDDR1 C-terminus (Cell), anti-PY, or anti-actin. (D) HEK293 cells were transiently transfected as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti-DDR1 ectodomain and anti-actin antibodies. For the phosphotyrosine blot, cells treated with collagen for 1 h were used. The relative intensities of shed DDR1 are shown at the bottom of the top panel. (E) HEK293 cells were transfected with N-terminally FLAG-tagged DDR1 mutants as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were analyzed by Western blotting as in D.

Mentions: There are two potential mechanisms to explain collagen-induced shedding of DDR1. First, collagen-induced DDR1 signaling may trigger the functional activation of ADAM10 (Vogel, 2002); second, the orientation of DDR1 may be altered upon collagen binding, which allows ADAM10 to cleave DDR1. We first investigated whether collagen-induced DDR1 signaling plays a role. We used a kinase-dead mutant of DDR1 (DDR1-KD) and a cytoplasmic domain deletion mutant (DDR1∆C), both of which are not able to transmit collagen signals (Figure 6A). We confirmed that those mutants are expressed on the cell surface in a similar manner to wild-type (WT) DDR1 by a surface biotinylation assay (Supplemental Figure S4A). As shown in Figure 6B, upon collagen stimulation, the ectodomain of DDR1-KD and DDR1∆C was shed in an efficient manner. We also examined the effect of a tyrosine kinase inhibitor, dasatinib, which inhibits DDR1 with an IC50 of 1.35 nM (Day et al., 2008). As shown in Figure 6C, dasatinib treatment at 50 nM did not influence DDR1 shedding, whereas it completely inhibited phosphorylation of DDR1 upon collagen stimulation. These data strongly suggest that collagen-induced DDR1 shedding is not dependent on DDR1 signaling.


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)

Collagen binding, but not DDR1 phosphorylation, is required for DDR1 shedding. (A) Schematic representation of mutant DDR1 constructs used in the experiments. DD, discoidin-homology domain; ∆C, cytoplasmic domain-deleted; DLD, discoidin-like domain; FLAG, FLAG tag (DYKDDDDK); HA, HA tag (YPYDVPDYA); JM, juxtamembrane region; KD, kinase dead; S, signal peptide; TM, transmembrane domain; TKD, tyrosine kinase domain. (B) HEK293 cells expressing DDR1-WT (WT), DDR1-KD (KD), or DDR1∆C (∆C) were treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1 ectodomain (Med and Cell), anti–phosphotyrosine 4G10 (PY), or anti-actin. Asterisk indicates tyrosine-phosphorylated proteins other than DDR1. (C) HEK293 cells expressing DDR1-WT were treated with collagen I in the presence or absence of 50 nM dasatinib or vehicle control (DMSO) for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), antixDDR1 C-terminus (Cell), anti-PY, or anti-actin. (D) HEK293 cells were transiently transfected as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti-DDR1 ectodomain and anti-actin antibodies. For the phosphotyrosine blot, cells treated with collagen for 1 h were used. The relative intensities of shed DDR1 are shown at the bottom of the top panel. (E) HEK293 cells were transfected with N-terminally FLAG-tagged DDR1 mutants as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were analyzed by Western blotting as in D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325837&req=5

Figure 6: Collagen binding, but not DDR1 phosphorylation, is required for DDR1 shedding. (A) Schematic representation of mutant DDR1 constructs used in the experiments. DD, discoidin-homology domain; ∆C, cytoplasmic domain-deleted; DLD, discoidin-like domain; FLAG, FLAG tag (DYKDDDDK); HA, HA tag (YPYDVPDYA); JM, juxtamembrane region; KD, kinase dead; S, signal peptide; TM, transmembrane domain; TKD, tyrosine kinase domain. (B) HEK293 cells expressing DDR1-WT (WT), DDR1-KD (KD), or DDR1∆C (∆C) were treated with collagen I for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti-DDR1 ectodomain (Med and Cell), anti–phosphotyrosine 4G10 (PY), or anti-actin. Asterisk indicates tyrosine-phosphorylated proteins other than DDR1. (C) HEK293 cells expressing DDR1-WT were treated with collagen I in the presence or absence of 50 nM dasatinib or vehicle control (DMSO) for 24 h. Conditioned media and cell lysates were analyzed by Western blotting using anti–DDR1 ectodomain (Med), antixDDR1 C-terminus (Cell), anti-PY, or anti-actin. (D) HEK293 cells were transiently transfected as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were subjected to Western blotting using anti-DDR1 ectodomain and anti-actin antibodies. For the phosphotyrosine blot, cells treated with collagen for 1 h were used. The relative intensities of shed DDR1 are shown at the bottom of the top panel. (E) HEK293 cells were transfected with N-terminally FLAG-tagged DDR1 mutants as indicated and treated with collagen for 24 h. Conditioned media and cell lysates were analyzed by Western blotting as in D.
Mentions: There are two potential mechanisms to explain collagen-induced shedding of DDR1. First, collagen-induced DDR1 signaling may trigger the functional activation of ADAM10 (Vogel, 2002); second, the orientation of DDR1 may be altered upon collagen binding, which allows ADAM10 to cleave DDR1. We first investigated whether collagen-induced DDR1 signaling plays a role. We used a kinase-dead mutant of DDR1 (DDR1-KD) and a cytoplasmic domain deletion mutant (DDR1∆C), both of which are not able to transmit collagen signals (Figure 6A). We confirmed that those mutants are expressed on the cell surface in a similar manner to wild-type (WT) DDR1 by a surface biotinylation assay (Supplemental Figure S4A). As shown in Figure 6B, upon collagen stimulation, the ectodomain of DDR1-KD and DDR1∆C was shed in an efficient manner. We also examined the effect of a tyrosine kinase inhibitor, dasatinib, which inhibits DDR1 with an IC50 of 1.35 nM (Day et al., 2008). As shown in Figure 6C, dasatinib treatment at 50 nM did not influence DDR1 shedding, whereas it completely inhibited phosphorylation of DDR1 upon collagen stimulation. These data strongly suggest that collagen-induced DDR1 shedding is not dependent on DDR1 signaling.

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