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. 2010 Jun 1;19(11):2239-50.
doi: 10.1093/hmg/ddq103. Epub 2010 Mar 10.

Trafficking Defects and Loss of Ligand Binding Are the Underlying Causes of All Reported DDR2 Missense Mutations Found in SMED-SL Patients

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Trafficking Defects and Loss of Ligand Binding Are the Underlying Causes of All Reported DDR2 Missense Mutations Found in SMED-SL Patients

Bassam R Ali et al. Hum Mol Genet. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
Schematic domain structure of homodimeric DDR2. The extracellular domain consists of a collagen-binding discoidin domain, followed by a so-called stalk region. The intracellular domain contains a large cytosolic juxtamembrane domain in addition to the C-terminal tyrosine kinase domain. The position of disease-causing missense mutations is shown at the left.
Figure 2.
Figure 2.
Chest and hand radiographs of cases 1 and 2. (A) Chest radiograph of case 1 at 9.5 years. Narrow thorax with short broad ribs, more at the lateral and anterior aspects (thin arrows) than at the costo-vertebral joints. Note very few calcifications at the costal cartilage on the right side (arrow heads). Note also irregularities at the humeral meta/epiphyses with calcifications (thick arrows). (B) Radiograph of the left hand (dorso-volar projection) of case 2 at 8 years of age showing short and broad tubular bones with typical diaphyseal constriction (medium arrow). The metacarpal bones are narrower at the proximal end giving the appearance of drumsticks (thick arrow). There is some bone-within-bone appearance of the phalanges (medium arrow). The distal phalanges are triangular in shape. The epiphyses of the phalanges appear irregular and cone-shaped (arrow head). Small and irregular precocious carpal bones. The distal metaphyses of the radius and ulna is broad and irregular with irregular trabecular structure. The distal ulnar epiphysis is triangular in shape (thin arrow).
Figure 3.
Figure 3.
Comparison of subcellular localization of DDR2 wild-type and SMED-SL patient mutant variants. HeLa cells were transiently co-transfected with plasmids encoding the indicated HA-tagged DDR2 protein and EGFP-tagged H-Ras, fixed and stained with anti-HA antibodies as described in Materials and Methods. (A), (D), (G), (J) and (M) show the distribution of over-expressed HA-tagged DDR2 proteins. (B), (E), (H), (K) and (N) show the distribution of over-expressed EGFP-tagged H-Ras, which is predominantly localized to the plasma membrane, while (C), (F), (I), (L) and (O) show the extent of co-localization of DDR2 proteins with EGFP-H-Ras.
Figure 4.
Figure 4.
Comparison of the intracellular localization of DDR2 wild-type and SMED-SL patient mutant variants with the ER marker. HeLa cells were transiently transfected with plasmids encoding the indicated HA-tagged DDR2 protein, fixed and stained with antibodies against the HA-tag (monoclonal) and calnexin (polyclonal) as described in Materials and Methods. (A), (D), (G), (J) and (M) show the distribution of over-expressed HA-tagged DDR2 proteins. (B), (E), (H), (K) and (N) show the distribution of calnexin, which is predominantly localized to the ER, while (C), (F), (I), (L) and (O) show the extent of co-localization of DDR2 proteins with calnexin.
Figure 5.
Figure 5.
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.
Figure 6.
Figure 6.
Loss of collagen-induced signaling despite normal cellular trafficking of E113K-DDR2. Untagged full-length DDR2 wild-type or E113K-DDR2 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 for 90 min at 37°C with rat tail collagen I at the indicated concentrations (in μg/ml). Cell lysates were analyzed 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.
Figure 7.
Figure 7.
Loss of collagen-binding function of E113K-DDR2. (A) SDS–polyacrylamide gel electrophoresis of purified WT-DDR2-Fc, E113K-DDR2-Fc and W52A-DDR2-Fc. A Coomassie-blue-stained gel (7.5% gel) is shown. The positions of molecular weight markers (in kDa) are indicated. (B and C) Solid phase binding assays with recombinant wild-type DDR2-Fc, E113K-DDR2-Fc or W52A-DDR2-Fc. Recombinant DDR2 ectodomain proteins were added for 3 h at room temperature to 96-well plates coated with collagen I (B) or collagen II (C) at 10 µg/ml. Shown are the means ± SEM of three independent experiments, each performed in duplicates.
Figure 8.
Figure 8.
Cartoon drawing of the DDR2 discoidin domain (selected side chains: W52, cyan; R105, blue; E113, red) bound to a collagen peptide (selected side chains: M21 and O24 of leading chain, F23 of middle chain). Hydrogen bonds involving E113 are shown as dashed black lines. The Figure was prepared using the coordinates of PDB entry 2WUH (6).

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References

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