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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.

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.

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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.
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DDQ103F3: 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.

Mentions: We hypothesized that most of the missense mutations in DDR2 (Fig. 1) will result in the retention of the mutated protein in the ER. This assumption was based on the relatedness of DDR2 to ROR2 and our findings that all the pathogenic missense mutations in ROR2 have been shown to be retained in the ER (15,16). Therefore, we generated all the missense mutations found in SMED-SL patients including those reported by Bargal et al. (11) in a mammalian expression system using site-directed mutagenesis and expressed the proteins in HeLa cells as described in the Materials and Methods. Confocal microscopy imaging revealed that the C-terminally HA-tagged wild-type DDR2 protein was primarily localized to the cell surface and colocalized with the plasma membrane marker GFP-h-Ras (Fig. 3A–C). We also observed some intracellular staining of the wild-type protein which presumably represents the newly synthesized protein that is in transit to the cell surface. Similarly, the p.E113K mutant was located to the periphery of the cell and colocalized with GFP-h-Ras (Fig. 3D–F). In contrast, confocal imaging of p.T713I, p.I726R and p.R752C mutants revealed an intracellular and reticular localization of the proteins, which is clearly different from that of the wild-type protein, with no colocalization with the plasma membrane marker (Fig. 3G–O). This intracellular staining is reminiscent of ER staining and we therefore co-stained cells expressing the wild-type and mutant proteins with a well established ER marker (calnexin). As shown in Figure 4A–F, the wild-type protein and p.E113K mutant showed distinct and different localizations from that of calnexin, confirming plasma membrane locations. On the other hand, the three intracellular missense mutants (p.T713I, p.I726R and p.R752C) showed a high degree of colocalization with the ER marker (Fig. 4G–O) indicating ER localization. Further biochemical studies (the following sections) confirmed the ER localization of the three proteins.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDQ103F3: 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.
Mentions: We hypothesized that most of the missense mutations in DDR2 (Fig. 1) will result in the retention of the mutated protein in the ER. This assumption was based on the relatedness of DDR2 to ROR2 and our findings that all the pathogenic missense mutations in ROR2 have been shown to be retained in the ER (15,16). Therefore, we generated all the missense mutations found in SMED-SL patients including those reported by Bargal et al. (11) in a mammalian expression system using site-directed mutagenesis and expressed the proteins in HeLa cells as described in the Materials and Methods. Confocal microscopy imaging revealed that the C-terminally HA-tagged wild-type DDR2 protein was primarily localized to the cell surface and colocalized with the plasma membrane marker GFP-h-Ras (Fig. 3A–C). We also observed some intracellular staining of the wild-type protein which presumably represents the newly synthesized protein that is in transit to the cell surface. Similarly, the p.E113K mutant was located to the periphery of the cell and colocalized with GFP-h-Ras (Fig. 3D–F). In contrast, confocal imaging of p.T713I, p.I726R and p.R752C mutants revealed an intracellular and reticular localization of the proteins, which is clearly different from that of the wild-type protein, with no colocalization with the plasma membrane marker (Fig. 3G–O). This intracellular staining is reminiscent of ER staining and we therefore co-stained cells expressing the wild-type and mutant proteins with a well established ER marker (calnexin). As shown in Figure 4A–F, the wild-type protein and p.E113K mutant showed distinct and different localizations from that of calnexin, confirming plasma membrane locations. On the other hand, the three intracellular missense mutants (p.T713I, p.I726R and p.R752C) showed a high degree of colocalization with the ER marker (Fig. 4G–O) indicating ER localization. Further biochemical studies (the following sections) confirmed the ER localization of the three proteins.

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