Bowman Amber L, Catino Dawn H, Strong John C, Randall William R, Kontrogianni-Konstantopoulos Aikaterini, Bloch Robert J
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Mol Biol Cell. 2008 Sep;19(9):3782-92. doi: 10.1091/mbc.e08-03-0237. Epub 2008 Jun 25.
Obscurin is an approximately 800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including alpha-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 microM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.
obscurin是一种约800 kDa的蛋白质，由结构域和信号结构域组成，可在横纹肌中组织收缩结构。我们研究了obscurin的Rho-GEF结构域，以了解其在形态发生和信号传导中的作用。我们使用该结构域的腺病毒过表达，结合超微结构和免疫荧光方法，来检查其对成熟肌原纤维的影响。我们报告说，Rho-GEF结构域的过表达特异性抑制肌联蛋白掺入发育中的Z盘，破坏Z盘和Z/I连接的结构，并改变A/I连接的特征。其他肌节标记物，包括α-辅肌动蛋白的组织不受影响。我们鉴定出Ran结合蛋白9（RanBP9）是Rho-GEF结构域的一种新型配体，并表明结合是特异性的，表观结合亲和力为1.9 microM。RanBP9结合区域的过表达也破坏了肌联蛋白掺入发育中的Z盘。肌原纤维形成过程中的免疫荧光定位表明，Rho-GEF结构域在RanBP9之前组装到肌节中，RanBP9首先出现在肌核中，随后在发育过程中转移到肌浆中，在那里它与obscurin共定位。Rho-GEF结构域及其在RanBP9上的结合区域都直接与位于Z盘两侧的肌联蛋白的N端免疫球蛋白结构域结合。我们的结果表明，Rho-GEF结构域与RanBP9相互作用，并且两者都可以与肌联蛋白的N端区域相互作用，以影响Z盘和A/I连接的形成。
