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Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling.

Lee HK, Ji S, Park SJ, Choung HW, Choi Y, Lee HJ, Park SY, Park JC - J. Biol. Chem. (2015)

Bottom Line: Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown.ODAM-mediated RhoA signaling resulted in actin filament rearrangement.These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Oral Histology/Developmental Biology and.

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Related in: MedlinePlus

ODAM interacted with ARHGEF5 in ameloblasts.A, IP was performed using anti-ODAM antibody in ALCs. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 antibody. B, ALCs were cotransfected with HA-ODAM and ARHGEF5 constructs. IP was performed using anti-HA or ARHGEF5 antibodies. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 or HA antibodies. C, mapping of the ODAM domain required for interaction with ARHGEF5. FLAG-ODAM mutants were expressed in ALCs transfected with ARHGEF5. The interaction was evaluated by IP using the anti-FLAG antibody, followed by Western blotting using anti-ARHGEF5 antibody. D, ALCs were transfected with the FLAG-ARHGEF5 mutant containing only the SH domain (amino acids 1489–1581). His pulldown assays were performed with cells expressing the ARHGEF5 SH domain. The ARHGEF5 interaction was determined by pulldown using the His-ODAM C-terminal mutant. Interactions were detected by Western blotting (WB) using an antibody specific for the FLAG tag expressed by the ARHGEF5 mutant. E, GFP-tagged ODAM and FLAG-tagged ARHGEF5 constructs were transfected into ALCs. Exogenous ARHGEF5 was immunostained using the anti-FLAG antibody, and GFP-ODAM was detected by immunofluorescence. Scale bars = 20 μm.
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Figure 3: ODAM interacted with ARHGEF5 in ameloblasts.A, IP was performed using anti-ODAM antibody in ALCs. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 antibody. B, ALCs were cotransfected with HA-ODAM and ARHGEF5 constructs. IP was performed using anti-HA or ARHGEF5 antibodies. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 or HA antibodies. C, mapping of the ODAM domain required for interaction with ARHGEF5. FLAG-ODAM mutants were expressed in ALCs transfected with ARHGEF5. The interaction was evaluated by IP using the anti-FLAG antibody, followed by Western blotting using anti-ARHGEF5 antibody. D, ALCs were transfected with the FLAG-ARHGEF5 mutant containing only the SH domain (amino acids 1489–1581). His pulldown assays were performed with cells expressing the ARHGEF5 SH domain. The ARHGEF5 interaction was determined by pulldown using the His-ODAM C-terminal mutant. Interactions were detected by Western blotting (WB) using an antibody specific for the FLAG tag expressed by the ARHGEF5 mutant. E, GFP-tagged ODAM and FLAG-tagged ARHGEF5 constructs were transfected into ALCs. Exogenous ARHGEF5 was immunostained using the anti-FLAG antibody, and GFP-ODAM was detected by immunofluorescence. Scale bars = 20 μm.

Mentions: In our previous study, ARHGEF5 was identified as an ODAM-interacting protein by protoarray analysis (22). In immunoprecipitation (IP) assay, ODAM also showed endogenous interaction with ARHGEF5 in ALCs (Fig. 3A). To confirm whether ODAM could interact with ARHGEF5, ALCs were cotransfected with ARHGEF5and HA-tagged ODAM constructs for IP assay. The results demonstrated the interaction of ODAM with ARHGEF5 (Fig. 3B). IP with the FLAG antibody followed by blotting with the ARHEGF5 antibody indicated that amino acids 127–279 of ODAM affected the interaction between ODAM and ARHGEF5 (Fig. 3C). Pulldown assays also showed a direct interaction between these two proteins (Fig. 3D). Immunofluorescence microscopy revealed that the majority of GFP-tagged ODAM and FLAG-tagged ARHGEF5 proteins colocalized to the periphery of ALCs (Fig. 3E). Overall, these data suggest that the interaction between the C terminus of ODAM and the SH domain of ARHGEF5 occurs in the cell periphery of ameloblasts.


Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling.

Lee HK, Ji S, Park SJ, Choung HW, Choi Y, Lee HJ, Park SY, Park JC - J. Biol. Chem. (2015)

ODAM interacted with ARHGEF5 in ameloblasts.A, IP was performed using anti-ODAM antibody in ALCs. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 antibody. B, ALCs were cotransfected with HA-ODAM and ARHGEF5 constructs. IP was performed using anti-HA or ARHGEF5 antibodies. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 or HA antibodies. C, mapping of the ODAM domain required for interaction with ARHGEF5. FLAG-ODAM mutants were expressed in ALCs transfected with ARHGEF5. The interaction was evaluated by IP using the anti-FLAG antibody, followed by Western blotting using anti-ARHGEF5 antibody. D, ALCs were transfected with the FLAG-ARHGEF5 mutant containing only the SH domain (amino acids 1489–1581). His pulldown assays were performed with cells expressing the ARHGEF5 SH domain. The ARHGEF5 interaction was determined by pulldown using the His-ODAM C-terminal mutant. Interactions were detected by Western blotting (WB) using an antibody specific for the FLAG tag expressed by the ARHGEF5 mutant. E, GFP-tagged ODAM and FLAG-tagged ARHGEF5 constructs were transfected into ALCs. Exogenous ARHGEF5 was immunostained using the anti-FLAG antibody, and GFP-ODAM was detected by immunofluorescence. Scale bars = 20 μm.
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Related In: Results  -  Collection

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Figure 3: ODAM interacted with ARHGEF5 in ameloblasts.A, IP was performed using anti-ODAM antibody in ALCs. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 antibody. B, ALCs were cotransfected with HA-ODAM and ARHGEF5 constructs. IP was performed using anti-HA or ARHGEF5 antibodies. Precipitated proteins were visualized by Western blotting using anti-ARHGEF5 or HA antibodies. C, mapping of the ODAM domain required for interaction with ARHGEF5. FLAG-ODAM mutants were expressed in ALCs transfected with ARHGEF5. The interaction was evaluated by IP using the anti-FLAG antibody, followed by Western blotting using anti-ARHGEF5 antibody. D, ALCs were transfected with the FLAG-ARHGEF5 mutant containing only the SH domain (amino acids 1489–1581). His pulldown assays were performed with cells expressing the ARHGEF5 SH domain. The ARHGEF5 interaction was determined by pulldown using the His-ODAM C-terminal mutant. Interactions were detected by Western blotting (WB) using an antibody specific for the FLAG tag expressed by the ARHGEF5 mutant. E, GFP-tagged ODAM and FLAG-tagged ARHGEF5 constructs were transfected into ALCs. Exogenous ARHGEF5 was immunostained using the anti-FLAG antibody, and GFP-ODAM was detected by immunofluorescence. Scale bars = 20 μm.
Mentions: In our previous study, ARHGEF5 was identified as an ODAM-interacting protein by protoarray analysis (22). In immunoprecipitation (IP) assay, ODAM also showed endogenous interaction with ARHGEF5 in ALCs (Fig. 3A). To confirm whether ODAM could interact with ARHGEF5, ALCs were cotransfected with ARHGEF5and HA-tagged ODAM constructs for IP assay. The results demonstrated the interaction of ODAM with ARHGEF5 (Fig. 3B). IP with the FLAG antibody followed by blotting with the ARHEGF5 antibody indicated that amino acids 127–279 of ODAM affected the interaction between ODAM and ARHGEF5 (Fig. 3C). Pulldown assays also showed a direct interaction between these two proteins (Fig. 3D). Immunofluorescence microscopy revealed that the majority of GFP-tagged ODAM and FLAG-tagged ARHGEF5 proteins colocalized to the periphery of ALCs (Fig. 3E). Overall, these data suggest that the interaction between the C terminus of ODAM and the SH domain of ARHGEF5 occurs in the cell periphery of ameloblasts.

Bottom Line: Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown.ODAM-mediated RhoA signaling resulted in actin filament rearrangement.These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Oral Histology/Developmental Biology and.

Show MeSH
Related in: MedlinePlus