In striated muscles, the large scaffolding protein obscurin and a small SR-integral membrane protein sAnk1.5 control the retention of longitudinal SR across the sarcomere. How a complex of these proteins facilitates localization of longitudinal SR has yet to be resolved, but we hypothesize that obscurin interacts with a complex of sAnk1.5 proteins. To begin to address this hypothesis, we demonstrate that sAnk1.5 interacts with itself and identify two domains mediating self-association. Specifically, we show by co-precipitation and FLIM-FRET analysis that sAnk1.5 and another small AnkR isoform (sAnk1.6) interact with themselves and each other. We demonstrate that obscurin interacts with a complex of sAnk1.5 proteins and that this complex formation is enhanced by obscurin-binding. Using FLIM-FRET analysis, we show that obscurin interacts with sAnk1.5 alone and with sAnk1.6 in the presence of sAnk1.5. We find that sAnk1.5 self-association is disrupted by mutagenesis of residues Arg64-Arg69, residues previously associated with obscurin-binding. Molecular modeling of two interacting sAnk1.5 monomers facilitated the identification of Gly31-Val36 as an additional site of interaction, which was subsequently corroborated by co-precipitation and FLIM-FRET analysis. In closing, these results support a model in which sAnk1.5 forms large oligomers that interact with obscurin to facilitate the retention of longitudinal SR throughout skeletal and cardiac myocytes.

中文翻译：

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小锚蛋白1亚型上的自缔合域的鉴定和表征。

在横纹肌中，大的支架蛋白obscurin和小的SR整合膜蛋白sAnk1.5控制整个肌小节的纵向SR保留。这些蛋白质的复合物如何促进纵向SR的定位尚待解决，但我们假设obscurin与sAnk1.5蛋白质的复合物相互作用。为了解决这个假设，我们证明了sAnk1.5与自身相互作用并确定了两个介导自我联想的域。具体而言，我们通过共沉淀和FLIM-FRET分析表明sAnk1.5和另一个小的AnkR亚型（sAnk1.6）彼此相互作用。我们证明，obscurin与sAnk1.5蛋白的复合物相互作用，并且该复合物的形成通过obscurin结合而增强。使用FLIM-FRET分析，我们显示了obscurin与sAnk1相互作用。sAnk1.5时，单独使用5和sAnk1.6。我们发现sAnk1.5自缔合被残基Arg64-Arg69诱变所破坏，残基Arg64-Arg69先前与暗抑素结合相关。两个相互作用的sAnk1.5单体的分子模型有助于将Gly31-Val36鉴定为另一个相互作用位点，随后通过共沉淀和FLIM-FRET分析证实了这一点。最后，这些结果支持了一个模型，其中sAnk1.5形成与obscurin相互作用的大型寡聚物，以促进纵向SR在整个骨骼肌和心肌细胞中的保留。5种单体有助于将Gly31-Val36鉴定为另一个相互作用位点，随后通过共沉淀和FLIM-FRET分析证实了这一点。最后，这些结果支持了一个模型，其中sAnk1.5形成与obscurin相互作用的大型寡聚物，以促进纵向SR在整个骨骼肌和心肌细胞中的保留。5种单体有助于将Gly31-Val36鉴定为另一个相互作用位点，随后通过共沉淀和FLIM-FRET分析证实了这一点。最后，这些结果支持了一个模型，其中sAnk1.5形成与obscurin相互作用的大型寡聚物，以促进纵向SR在整个骨骼肌和心肌细胞中的保留。