Limits...
Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients.

Ali BR, Xu H, Akawi NA, John A, Karuvantevida NS, Langer R, Al-Gazali L, Leitinger B - Hum. Mol. Genet. (2010)

Bottom Line: We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum.The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen.Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.

ABSTRACT
Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.

Show MeSH

Related in: MedlinePlus

Defective cellular trafficking causes loss of collagen-induced signaling for the SMED-SL patient mutant variants T713I-DDR2, I726R-DDR2 and R752C-DDR2. HA-tagged full-length DDR2 wild-type or mutant variants were transiently expressed in HEK293 cells. (A) Cell lysates were treated with Endoglycosidase H for 3 h at 37°C (H) or left untreated for 3 h at 37°C (−) and analyzed by SDS–PAGE and western blotting. The blot was probed with polyclonal anti-DDR2 antibodies. (B) Cells were stimulated with 10 µg/ml of rat tail collagen I (+) or 1 mm acetic acid (−) for 90 min at 37°C. Cell lysates were analysed by SDS–PAGE and western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower blot). The positions of molecular markers (in kDa) are indicated. The experiments were carried out three times with very similar results.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2865377&req=5

DDQ103F5: Defective cellular trafficking causes loss of collagen-induced signaling for the SMED-SL patient mutant variants T713I-DDR2, I726R-DDR2 and R752C-DDR2. HA-tagged full-length DDR2 wild-type or mutant variants were transiently expressed in HEK293 cells. (A) Cell lysates were treated with Endoglycosidase H for 3 h at 37°C (H) or left untreated for 3 h at 37°C (−) and analyzed by SDS–PAGE and western blotting. The blot was probed with polyclonal anti-DDR2 antibodies. (B) Cells were stimulated with 10 µg/ml of rat tail collagen I (+) or 1 mm acetic acid (−) for 90 min at 37°C. Cell lysates were analysed by SDS–PAGE and western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower blot). The positions of molecular markers (in kDa) are indicated. The experiments were carried out three times with very similar results.

Mentions: The first step in collagen-induced DDR2 transmembrane signaling is activation of DDR2, which manifests itself in autophosphorylation of cytoplasmic tyrosine residues (1,2). To analyze whether SMED-SL disease mutants could be activated by binding to collagen, we transiently expressed the mutants and wild-type DDR2 in HEK293 cells. HEK293 cells were chosen for these experiments, as DDR autophosphorylation in these cells can be easily monitored by western blotting of cell lysates (1,21). In transfected HEK293 cells, wild-type DDR2 is usually observed as a mixture of three (sometimes four) molecular weight forms between ∼125 and 130 kDa (5,21,22), with the upper forms representing complex glycosylated mature forms. Unlike wild-type DDR2, however, for which the expected molecular weight forms were detected (Fig. 5A), the SMED-SL mutants p.T713I, p.I726R and p.R752C were detected predominantly as the lowest molecular weight species (Fig. 5A), with mutants p.T713I and p.I726R displaying a small amount of the middle forms. None of these mutants displayed the highest molecular weight form of DDR2. In addition, Endo H treatment of cell lysates expressing wild-type or mutant DDR2 showed that the predominant forms of T713I-DDR2, I726R-DDR2 and R752C-DDR2 were sensitive to Endo H digestion, as was the lowest molecular weight form of wild-type DDR2 (Fig. 5A), suggesting that these molecular weight species represent immature biosynthetic precursors of DDR2. This is in agreement with the protein localization data in HeLa cells, presented in Figures 3 and 4. The predominant forms of wild-type DDR2, however, were the upper molecular weight species, which were largely absent in the mutant receptors. These forms were Endo H resistant and thus represent the complex glycosylated mature forms of DDR2.


Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients.

Ali BR, Xu H, Akawi NA, John A, Karuvantevida NS, Langer R, Al-Gazali L, Leitinger B - Hum. Mol. Genet. (2010)

Defective cellular trafficking causes loss of collagen-induced signaling for the SMED-SL patient mutant variants T713I-DDR2, I726R-DDR2 and R752C-DDR2. HA-tagged full-length DDR2 wild-type or mutant variants were transiently expressed in HEK293 cells. (A) Cell lysates were treated with Endoglycosidase H for 3 h at 37°C (H) or left untreated for 3 h at 37°C (−) and analyzed by SDS–PAGE and western blotting. The blot was probed with polyclonal anti-DDR2 antibodies. (B) Cells were stimulated with 10 µg/ml of rat tail collagen I (+) or 1 mm acetic acid (−) for 90 min at 37°C. Cell lysates were analysed by SDS–PAGE and western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower blot). The positions of molecular markers (in kDa) are indicated. The experiments were carried out three times with very similar results.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2865377&req=5

DDQ103F5: Defective cellular trafficking causes loss of collagen-induced signaling for the SMED-SL patient mutant variants T713I-DDR2, I726R-DDR2 and R752C-DDR2. HA-tagged full-length DDR2 wild-type or mutant variants were transiently expressed in HEK293 cells. (A) Cell lysates were treated with Endoglycosidase H for 3 h at 37°C (H) or left untreated for 3 h at 37°C (−) and analyzed by SDS–PAGE and western blotting. The blot was probed with polyclonal anti-DDR2 antibodies. (B) Cells were stimulated with 10 µg/ml of rat tail collagen I (+) or 1 mm acetic acid (−) for 90 min at 37°C. Cell lysates were analysed by SDS–PAGE and western blotting. The blots were probed with anti-phosphotyrosine (anti-PY) monoclonal antibody 4G10 (upper blot) or polyclonal anti-DDR2 antibodies (lower blot). The positions of molecular markers (in kDa) are indicated. The experiments were carried out three times with very similar results.
Mentions: The first step in collagen-induced DDR2 transmembrane signaling is activation of DDR2, which manifests itself in autophosphorylation of cytoplasmic tyrosine residues (1,2). To analyze whether SMED-SL disease mutants could be activated by binding to collagen, we transiently expressed the mutants and wild-type DDR2 in HEK293 cells. HEK293 cells were chosen for these experiments, as DDR autophosphorylation in these cells can be easily monitored by western blotting of cell lysates (1,21). In transfected HEK293 cells, wild-type DDR2 is usually observed as a mixture of three (sometimes four) molecular weight forms between ∼125 and 130 kDa (5,21,22), with the upper forms representing complex glycosylated mature forms. Unlike wild-type DDR2, however, for which the expected molecular weight forms were detected (Fig. 5A), the SMED-SL mutants p.T713I, p.I726R and p.R752C were detected predominantly as the lowest molecular weight species (Fig. 5A), with mutants p.T713I and p.I726R displaying a small amount of the middle forms. None of these mutants displayed the highest molecular weight form of DDR2. In addition, Endo H treatment of cell lysates expressing wild-type or mutant DDR2 showed that the predominant forms of T713I-DDR2, I726R-DDR2 and R752C-DDR2 were sensitive to Endo H digestion, as was the lowest molecular weight form of wild-type DDR2 (Fig. 5A), suggesting that these molecular weight species represent immature biosynthetic precursors of DDR2. This is in agreement with the protein localization data in HeLa cells, presented in Figures 3 and 4. The predominant forms of wild-type DDR2, however, were the upper molecular weight species, which were largely absent in the mutant receptors. These forms were Endo H resistant and thus represent the complex glycosylated mature forms of DDR2.

Bottom Line: We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum.The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen.Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.

ABSTRACT
Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.

Show MeSH
Related in: MedlinePlus