Gimona M, Mital R
Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
J Cell Sci. 1998 Jul;111 ( Pt 13):1813-21. doi: 10.1242/jcs.111.13.1813.
Calponins have been implicated in the regulation of actomyosin interactions in smooth muscle cells, cytoskeletal organisation in nonmuscle cells, and the control of neurite outgrowth. Domains homologous to the amino-terminal region of calponin have been identified in a variety of actin cross-linking proteins and signal transduction molecules, and by inference these 'calponin homology (CH) domains' have been assumed to participate in actin binding. We here report on the actin binding activities of the subdomains of the calponin molecule. All three mammalian isoforms of calponin (basic h1, neutral h2 and acidic) possess a single CH domain at their amino terminus as well as three tandem repeats proximal to the carboxyl terminus. Calponin h2 differs, however, from h1 in lacking a consensus actin-binding motif in the region 142-163, between the CH domain and the tandem repeats, which in h1 calponin can be chemically cross-linked to actin. Despite the absence of this consensus actin-binding motif, recombinant full-length h2 calponin co-sediments in vitro with F-actin, suggesting the presence of another binding site in the molecule. It could be shown that this binding site resides in the C-terminal tandem repeats and not in the CH domain. Thus, constructs of h2 calponin bearing partial or complete deletions of the triple repeated sequences failed to co-localise with actin stress fibres despite the presence of a CH domain. Deletion of the acidic carboxyl terminus, beyond the repeats, increased actin binding, suggesting that the carboxy-terminal tail may modulate actin association. Results obtained from transient transfections of amino- and carboxy-terminal truncations in h1 calponin were consistent with the established location of the actin binding motif outside and carboxy-terminal to the CH domain, and confirm that the presence of a single CH domain alone is neither sufficient nor necessary to mediate actin binding. Instead, the carboxy-terminal tandem repeats of h1 and h2 calponin are shown to harbour a second, independent actin binding motif.
钙调蛋白与平滑肌细胞中肌动球蛋白相互作用的调节、非肌肉细胞中细胞骨架的组织以及神经突生长的控制有关。在多种肌动蛋白交联蛋白和信号转导分子中已鉴定出与钙调蛋白氨基末端区域同源的结构域，据此推测这些“钙调蛋白同源（CH）结构域”参与肌动蛋白结合。我们在此报告钙调蛋白分子亚结构域的肌动蛋白结合活性。钙调蛋白的所有三种哺乳动物同工型（碱性h1、中性h2和酸性）在其氨基末端都有一个单一的CH结构域，并且在羧基末端附近有三个串联重复序列。然而，钙调蛋白h2与h1的不同之处在于，在CH结构域和串联重复序列之间的142 - 163区域缺乏一个一致的肌动蛋白结合基序，在h1钙调蛋白中该区域可以与肌动蛋白化学交联。尽管没有这个一致的肌动蛋白结合基序，但重组全长h2钙调蛋白在体外与F - 肌动蛋白共沉降，表明该分子中存在另一个结合位点。可以证明这个结合位点位于C末端串联重复序列中，而不是在CH结构域中。因此，尽管存在CH结构域，但带有部分或完全缺失三重重复序列的h2钙调蛋白构建体未能与肌动蛋白应力纤维共定位。重复序列之外酸性羧基末端的缺失增加了肌动蛋白结合，表明羧基末端尾巴可能调节肌动蛋白结合。从h1钙调蛋白氨基末端和羧基末端截短体的瞬时转染获得的结果与已确定的肌动蛋白结合基序位于CH结构域之外和羧基末端的位置一致，并证实仅存在一个单一的CH结构域既不足以也不必要介导肌动蛋白结合。相反，h1和h2钙调蛋白的羧基末端串联重复序列显示含有第二个独立的肌动蛋白结合基序。
