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Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles.

Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V - J. Cell Biol. (2003)

Bottom Line: In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin.In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum.Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

ABSTRACT
Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

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Effect of ank1.5 on the localization of obscurin in NIH 3T3 cells. NIH 3T3 cells were transfected with the obscurin subclone A7 joined in frame with GFP, the full-length cDNA of ank1.5, ank1.7, and mutant ank1.5 cloned in pcDNA3 vectors. Cells were decorated with anti-myc monoclonal antibodies and detected by a Cy3-conjugated anti–mouse antibody (red). Panels show representative sections from the top to bottom of the cells at ∼0.25-μm intervals. Bar, 5 μm.
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fig4: Effect of ank1.5 on the localization of obscurin in NIH 3T3 cells. NIH 3T3 cells were transfected with the obscurin subclone A7 joined in frame with GFP, the full-length cDNA of ank1.5, ank1.7, and mutant ank1.5 cloned in pcDNA3 vectors. Cells were decorated with anti-myc monoclonal antibodies and detected by a Cy3-conjugated anti–mouse antibody (red). Panels show representative sections from the top to bottom of the cells at ∼0.25-μm intervals. Bar, 5 μm.

Mentions: To study at the cellular level the interaction between ank1.5 and the COOH-terminal region of obscurin, an expression vector encoding a fusion protein made of GFP followed by the obscurin subclone A7 (GFP–ObsA7) was transfected into NIH 3T3 cells in the absence or in the presence of expression vectors encoding ank1.5, ank1.7, or a mutant ank1.5 unable to bind to obscurin. In transfected cells, ank1.5 or ank1.7 presented a staining corresponding to that of the ER compartment of the cells (Fig. 4, B, E, and H). On the contrary, the GFP–ObsA7 protein, when expressed alone, displayed a diffused cytosolic localization (unpublished data). In cells cotransfected with ank1.5, the GFP–ObsA7 staining was redistributed almost completely to the same sites where ank1.5 was present (Fig. 4, A–C). The specificity of this interaction was confirmed by the lack of colocalization when GFP–ObsA7 was cotransfected with the mutated version of ank1.5, which is unable to interact with obscurin (Fig. 4, D–F). In addition, in agreement with previous results in yeast two-hybrid and pull-down experiments, no colocalization was observed when ank1.7 was transfected with the GFP–ObsA7 plasmid (Fig. 4, G–I).


Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles.

Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V - J. Cell Biol. (2003)

Effect of ank1.5 on the localization of obscurin in NIH 3T3 cells. NIH 3T3 cells were transfected with the obscurin subclone A7 joined in frame with GFP, the full-length cDNA of ank1.5, ank1.7, and mutant ank1.5 cloned in pcDNA3 vectors. Cells were decorated with anti-myc monoclonal antibodies and detected by a Cy3-conjugated anti–mouse antibody (red). Panels show representative sections from the top to bottom of the cells at ∼0.25-μm intervals. Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172649&req=5

fig4: Effect of ank1.5 on the localization of obscurin in NIH 3T3 cells. NIH 3T3 cells were transfected with the obscurin subclone A7 joined in frame with GFP, the full-length cDNA of ank1.5, ank1.7, and mutant ank1.5 cloned in pcDNA3 vectors. Cells were decorated with anti-myc monoclonal antibodies and detected by a Cy3-conjugated anti–mouse antibody (red). Panels show representative sections from the top to bottom of the cells at ∼0.25-μm intervals. Bar, 5 μm.
Mentions: To study at the cellular level the interaction between ank1.5 and the COOH-terminal region of obscurin, an expression vector encoding a fusion protein made of GFP followed by the obscurin subclone A7 (GFP–ObsA7) was transfected into NIH 3T3 cells in the absence or in the presence of expression vectors encoding ank1.5, ank1.7, or a mutant ank1.5 unable to bind to obscurin. In transfected cells, ank1.5 or ank1.7 presented a staining corresponding to that of the ER compartment of the cells (Fig. 4, B, E, and H). On the contrary, the GFP–ObsA7 protein, when expressed alone, displayed a diffused cytosolic localization (unpublished data). In cells cotransfected with ank1.5, the GFP–ObsA7 staining was redistributed almost completely to the same sites where ank1.5 was present (Fig. 4, A–C). The specificity of this interaction was confirmed by the lack of colocalization when GFP–ObsA7 was cotransfected with the mutated version of ank1.5, which is unable to interact with obscurin (Fig. 4, D–F). In addition, in agreement with previous results in yeast two-hybrid and pull-down experiments, no colocalization was observed when ank1.7 was transfected with the GFP–ObsA7 plasmid (Fig. 4, G–I).

Bottom Line: In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin.In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum.Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

ABSTRACT
Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils. In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

Show MeSH