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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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Reduced DDR1 shedding is associated with its sustained phosphorylation. (A) HEK293 stably expressing FLAG-tagged wild type (WT) or -6xD mutant DDR1 (6xD) were treated with collagen (20 μg/ml) for 1 h, excess collagen was then washed out, and cells were incubated further as indicated. RIPA lysates were collected at the indicated times and immunoprecipitated with anti-FLAG beads, followed by Western blotting with anti-DDR1 (Cell) and anti-PY antibodies. Shed DDR1 at each time point was analyzed by anti-DDR1 antibody (Med). Representative data from two independent experiments. (B) Relative intensities of phosphorylated DDR1 bands from A (PY). The intensities were standardized by the intensity of phosphorylated DDR1 in 1-h collagen-treated sample for each construct. (C) Relative band intensities of shed DDR1 in A (DDR1, Med). Relative intensities were normalized to the band intensity of shed DDR1-6xD at 0-h incubation time.
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Figure 8: Reduced DDR1 shedding is associated with its sustained phosphorylation. (A) HEK293 stably expressing FLAG-tagged wild type (WT) or -6xD mutant DDR1 (6xD) were treated with collagen (20 μg/ml) for 1 h, excess collagen was then washed out, and cells were incubated further as indicated. RIPA lysates were collected at the indicated times and immunoprecipitated with anti-FLAG beads, followed by Western blotting with anti-DDR1 (Cell) and anti-PY antibodies. Shed DDR1 at each time point was analyzed by anti-DDR1 antibody (Med). Representative data from two independent experiments. (B) Relative intensities of phosphorylated DDR1 bands from A (PY). The intensities were standardized by the intensity of phosphorylated DDR1 in 1-h collagen-treated sample for each construct. (C) Relative band intensities of shed DDR1 in A (DDR1, Med). Relative intensities were normalized to the band intensity of shed DDR1-6xD at 0-h incubation time.

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.


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)

Reduced DDR1 shedding is associated with its sustained phosphorylation. (A) HEK293 stably expressing FLAG-tagged wild type (WT) or -6xD mutant DDR1 (6xD) were treated with collagen (20 μg/ml) for 1 h, excess collagen was then washed out, and cells were incubated further as indicated. RIPA lysates were collected at the indicated times and immunoprecipitated with anti-FLAG beads, followed by Western blotting with anti-DDR1 (Cell) and anti-PY antibodies. Shed DDR1 at each time point was analyzed by anti-DDR1 antibody (Med). Representative data from two independent experiments. (B) Relative intensities of phosphorylated DDR1 bands from A (PY). The intensities were standardized by the intensity of phosphorylated DDR1 in 1-h collagen-treated sample for each construct. (C) Relative band intensities of shed DDR1 in A (DDR1, Med). Relative intensities were normalized to the band intensity of shed DDR1-6xD at 0-h incubation time.
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Figure 8: Reduced DDR1 shedding is associated with its sustained phosphorylation. (A) HEK293 stably expressing FLAG-tagged wild type (WT) or -6xD mutant DDR1 (6xD) were treated with collagen (20 μg/ml) for 1 h, excess collagen was then washed out, and cells were incubated further as indicated. RIPA lysates were collected at the indicated times and immunoprecipitated with anti-FLAG beads, followed by Western blotting with anti-DDR1 (Cell) and anti-PY antibodies. Shed DDR1 at each time point was analyzed by anti-DDR1 antibody (Med). Representative data from two independent experiments. (B) Relative intensities of phosphorylated DDR1 bands from A (PY). The intensities were standardized by the intensity of phosphorylated DDR1 in 1-h collagen-treated sample for each construct. (C) Relative band intensities of shed DDR1 in A (DDR1, Med). Relative intensities were normalized to the band intensity of shed DDR1-6xD at 0-h incubation time.
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.

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