Niessen Petra, Clément Sophie, Fontao Lionel, Chaponnier Christine, Teunissen Birgit, Rensen Sander, van Eys Guillaume, Gabbiani Giulio
Department of Molecular Genetics, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, 6200 MD Maastricht, The Netherlands.
Exp Cell Res. 2004 Jan 1;292(1):170-8. doi: 10.1016/j.yexcr.2003.09.005.
The two major isoforms of smoothelin (A and B) contain a calponin homology (CH) domain, colocalize with alpha-smooth muscle actin (alpha-SMA) in stress fibers and are only expressed in contractile smooth muscle cells (SMCs). Based on these findings, we hypothesized that smoothelins are involved in smooth muscle cell contraction, presumably via interaction with actin. The interaction between smoothelins and three different actin isoforms (alpha- and gamma-smooth muscle and alpha-skeletal actin [alpha-SKA]) was investigated using several in vitro assays. Smoothelin-B co-immunoprecipitated with alpha-smooth muscle actin from pig aorta extracts. In rat embryonic fibroblasts, transfected smoothelins-A and -B associated with stress fibers. In vitro dot blot assays, in which immobilized actin was overlaid with radio-labeled smoothelin, showed binding of smoothelin-A to actin filaments, but not to monomeric G-actin. A truncated smoothelin, containing the calponin homology domain, associated with stress fibers when transfected and bound to actin filaments in overlay, but to a lesser extent. ELISA results showed that the binding of smoothelin to actin has no significant isoform specificity. Our results indicate an interaction between smoothelin and actin filaments. Moreover, the calponin homology domain and its surrounding sequences appear to be sufficient to accomplish this interaction, although the presence of other domains is apparently necessary to facilitate and/or strengthen the binding to actin.
平滑肌动蛋白（Smoothelin）的两种主要亚型（A和B）包含一个钙调蛋白同源（CH）结构域，与α-平滑肌肌动蛋白（α-SMA）在应力纤维中共定位，且仅在收缩性平滑肌细胞（SMC）中表达。基于这些发现，我们推测平滑肌动蛋白参与平滑肌细胞收缩，可能是通过与肌动蛋白相互作用实现的。我们使用了几种体外试验研究了平滑肌动蛋白与三种不同肌动蛋白亚型（α-和γ-平滑肌肌动蛋白以及α-骨骼肌肌动蛋白[α-SKA]）之间的相互作用。平滑肌动蛋白-B与猪主动脉提取物中的α-平滑肌肌动蛋白进行了共免疫沉淀。在大鼠胚胎成纤维细胞中，转染的平滑肌动蛋白-A和-B与应力纤维相关联。在体外斑点印迹试验中，将固定化的肌动蛋白与放射性标记的平滑肌动蛋白叠加，结果显示平滑肌动蛋白-A与肌动蛋白丝结合，但不与单体G-肌动蛋白结合。一种截短的平滑肌动蛋白，包含钙调蛋白同源结构域，转染后与应力纤维相关联，并在叠加试验中与肌动蛋白丝结合，但程度较低。酶联免疫吸附测定（ELISA）结果表明，平滑肌动蛋白与肌动蛋白的结合没有明显的亚型特异性。我们的结果表明平滑肌动蛋白与肌动蛋白丝之间存在相互作用。此外，钙调蛋白同源结构域及其周围序列似乎足以实现这种相互作用，尽管其他结构域的存在显然有助于和/或加强与肌动蛋白的结合。
