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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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Association of ank1.5 with the COOH-terminal region of obscurin. (A) Two-hybrid screening with the cytosolic tail of ank1.5 (aa 22–155) as “bait” identified seven positive clones, corresponding to the COOH terminus of the obscurin protein. (B) Fragments of the obscurin clone A7 were tested for the ability to interact with the bait ank1.5 in the two-hybrid system and with a GST–ank1.5 fusion protein in pull-down experiments. Both experiments demonstrated that the region of obscurin between aa 6236 and 6353 is capable to interact with ank1.5. +, detectable activity; −, no detectable activity. (C) A second series of obscurin fragments was prepared to further restrict the sequence responsible for binding with ank1.5 to 25 aa of obscurin (aa 6236–6260). (D) Mutagenesis of the aa in the COOH terminus of obscurin (aa 6236–6260) capable of mediating binding to ank1.5 was performed and mutant proteins were tested against GST–ank1.5 in in vitro binding assays.
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fig1: Association of ank1.5 with the COOH-terminal region of obscurin. (A) Two-hybrid screening with the cytosolic tail of ank1.5 (aa 22–155) as “bait” identified seven positive clones, corresponding to the COOH terminus of the obscurin protein. (B) Fragments of the obscurin clone A7 were tested for the ability to interact with the bait ank1.5 in the two-hybrid system and with a GST–ank1.5 fusion protein in pull-down experiments. Both experiments demonstrated that the region of obscurin between aa 6236 and 6353 is capable to interact with ank1.5. +, detectable activity; −, no detectable activity. (C) A second series of obscurin fragments was prepared to further restrict the sequence responsible for binding with ank1.5 to 25 aa of obscurin (aa 6236–6260). (D) Mutagenesis of the aa in the COOH terminus of obscurin (aa 6236–6260) capable of mediating binding to ank1.5 was performed and mutant proteins were tested against GST–ank1.5 in in vitro binding assays.

Mentions: To identify potential interaction partners for short isoforms of the ank1 gene, a human skeletal muscle cDNA library was screened using the yeast two-hybrid technique and the cytoplasmic tail of the ank1.5 cDNA as bait. Screening of a total of 5 × 105 clones resulted in the identification of seven positive colonies (A7, A14, A19, A26, A36, A62, and A38). DNA sequencing revealed that all clones corresponded to overlapping fragments of obscurin, a giant muscle protein associated with the myofibrils (Bang et al., 2001; Young et al., 2001). Obscurin has a modular architecture containing multiple Ig-like domains, two fibronectin (FN3)-like domains, and a RhoGEF/PH domain followed at the COOH terminus by a nonmodular sequence (Fig. 1 A). Five of the clones identified in the two-hybrid screening (A7, A14, A19, A26, and A36) contained totally or partially the RhoGEF/PH and two Ig-like domains, whereas clones A38 and A62 contained only the nonmodular sequence at the COOH terminus (Fig. 1 A). None of these clones contained the kinase domains identified by Bang et al. (2001) and Russell et al. (2002). Because all clones can interact with ank1.5, we concluded that the interaction site was located in the nonmodular sequence at the COOH terminus of obscurin, downstream of aa 6235.


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)

Association of ank1.5 with the COOH-terminal region of obscurin. (A) Two-hybrid screening with the cytosolic tail of ank1.5 (aa 22–155) as “bait” identified seven positive clones, corresponding to the COOH terminus of the obscurin protein. (B) Fragments of the obscurin clone A7 were tested for the ability to interact with the bait ank1.5 in the two-hybrid system and with a GST–ank1.5 fusion protein in pull-down experiments. Both experiments demonstrated that the region of obscurin between aa 6236 and 6353 is capable to interact with ank1.5. +, detectable activity; −, no detectable activity. (C) A second series of obscurin fragments was prepared to further restrict the sequence responsible for binding with ank1.5 to 25 aa of obscurin (aa 6236–6260). (D) Mutagenesis of the aa in the COOH terminus of obscurin (aa 6236–6260) capable of mediating binding to ank1.5 was performed and mutant proteins were tested against GST–ank1.5 in in vitro binding assays.
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fig1: Association of ank1.5 with the COOH-terminal region of obscurin. (A) Two-hybrid screening with the cytosolic tail of ank1.5 (aa 22–155) as “bait” identified seven positive clones, corresponding to the COOH terminus of the obscurin protein. (B) Fragments of the obscurin clone A7 were tested for the ability to interact with the bait ank1.5 in the two-hybrid system and with a GST–ank1.5 fusion protein in pull-down experiments. Both experiments demonstrated that the region of obscurin between aa 6236 and 6353 is capable to interact with ank1.5. +, detectable activity; −, no detectable activity. (C) A second series of obscurin fragments was prepared to further restrict the sequence responsible for binding with ank1.5 to 25 aa of obscurin (aa 6236–6260). (D) Mutagenesis of the aa in the COOH terminus of obscurin (aa 6236–6260) capable of mediating binding to ank1.5 was performed and mutant proteins were tested against GST–ank1.5 in in vitro binding assays.
Mentions: To identify potential interaction partners for short isoforms of the ank1 gene, a human skeletal muscle cDNA library was screened using the yeast two-hybrid technique and the cytoplasmic tail of the ank1.5 cDNA as bait. Screening of a total of 5 × 105 clones resulted in the identification of seven positive colonies (A7, A14, A19, A26, A36, A62, and A38). DNA sequencing revealed that all clones corresponded to overlapping fragments of obscurin, a giant muscle protein associated with the myofibrils (Bang et al., 2001; Young et al., 2001). Obscurin has a modular architecture containing multiple Ig-like domains, two fibronectin (FN3)-like domains, and a RhoGEF/PH domain followed at the COOH terminus by a nonmodular sequence (Fig. 1 A). Five of the clones identified in the two-hybrid screening (A7, A14, A19, A26, and A36) contained totally or partially the RhoGEF/PH and two Ig-like domains, whereas clones A38 and A62 contained only the nonmodular sequence at the COOH terminus (Fig. 1 A). None of these clones contained the kinase domains identified by Bang et al. (2001) and Russell et al. (2002). Because all clones can interact with ank1.5, we concluded that the interaction site was located in the nonmodular sequence at the COOH terminus of obscurin, downstream of aa 6235.

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