Temm-Grove C J, Guo W, Helfman D M
Cold Spring Harbor Laboratory, NY 11724, USA.
Cell Motil Cytoskeleton. 1996;33(3):223-40. doi: 10.1002/(SICI)1097-0169(1996)33:3<223::AID-CM6>3.0.CO;2-B.
Previous studies have shown that three distinct genes encode six isoforms of tropomyosin (TM) in rat fibroblasts: the alpha gene encodes TM-2, TM-3, TM-5a, and TM-5b, the beta gene encodes TM-1, and the TM-4 gene encodes TM-4. Here we report the characterization of a cDNA clone encoding the most recent rat fibroblast TM to be identified, herein referred to as TM-5, that is the product of a fourth gene that is homologous to the human hTMnm gene, herein referred to as the rat slow-twitch alpha TM gene. The cDNA clone is approximately 1.7 kb and encodes a protein of 248 amino acids. Using two-dimensional gel electrophoresis, the TM-5 protein was found to co-migrate with fibroblast TM-5a and 5b. Comparison of the amino acid sequences of TM-5 to other fibroblast isoforms encoded by the alpha, beta, and TM-4 genes revealed a high degree of homology, although there were regions of divergence among the different isoforms. The gene encoding TM-5 is expressed in all tissues examined including skeletal muscle, stomach, heart, liver, kidney, uterus, spleen, brain, and diaphragm. However, Northern blot and RNase protection analyses revealed the presence of different mRNAs in fibroblasts, striated muscle (skeletal and diaphragm), and brain, which are expressed via alternative RNA splicing and the use of alternative promoters. The TM-5 protein was expressed in a bacterial system and tested for its ability to bind actin in vitro and in vivo. The apparent TM association constant (Ka) was taken as the free concentration at half saturation and was found to be 3 microM for TM-5 compared to 2 microM for TM-5b at an F-actin concentration of 42 microM. When fluorescently-labeled TM-5 was microinjected into living rat fibroblasts, it localized to the stress fibers and ruffles of the leading lamella. The coiled-coil interactions of TM-5 with other low and high molecular weight TM isoforms were studied. TM-5 and TM-4 were capable of dimerizing with each other as well as with other low molecular weight isoforms (TM-5a and TM-5b), but not with the HMW isoforms (TM-1, TM-2, and TM-3). In addition, TM-5a and TM-5b were unable to heterodimerize with each other. The implications of these results in understanding the role of TM diversity in cytoskeletal dynamics are discussed.
先前的研究表明，在大鼠成纤维细胞中，三个不同的基因编码六种原肌球蛋白（TM）同工型：α基因编码TM-2、TM-3、TM-5a和TM-5b，β基因编码TM-1，TM-4基因编码TM-4。在此，我们报告了一个cDNA克隆的特征，该克隆编码最近鉴定出的大鼠成纤维细胞TM，在此称为TM-5，它是与人类hTMnm基因同源的第四个基因的产物，在此称为大鼠慢肌αTM基因。该cDNA克隆约为1.7 kb，编码一个248个氨基酸的蛋白质。使用二维凝胶电泳，发现TM-5蛋白与成纤维细胞TM-5a和5b共迁移。将TM-5的氨基酸序列与由α、β和TM-4基因编码的其他成纤维细胞同工型进行比较，发现尽管不同同工型之间存在差异区域，但仍具有高度同源性。编码TM-5的基因在所有检测的组织中均有表达，包括骨骼肌、胃、心脏、肝脏、肾脏、子宫、脾脏、大脑和膈肌。然而，Northern印迹和核糖核酸酶保护分析显示，在成纤维细胞、横纹肌（骨骼肌和膈肌）和大脑中存在不同的mRNA，它们通过可变RNA剪接和使用可变启动子进行表达。TM-5蛋白在细菌系统中表达，并测试其在体外和体内结合肌动蛋白的能力。表观TM缔合常数（Ka）被视为半饱和时的游离浓度，发现在F-肌动蛋白浓度为42 μM时，TM-5的Ka为3 μM，而TM-5b为2 μM。当将荧光标记的TM-5显微注射到活的大鼠成纤维细胞中时，它定位于应力纤维和前缘片层的褶皱处。研究了TM-5与其他低分子量和高分子量TM同工型的卷曲螺旋相互作用。TM-5和TM-4能够彼此二聚化，也能与其他低分子量同工型（TM-5a和TM-5b）二聚化，但不能与高分子量同工型（TM-1、TM-2和TM-3）二聚化。此外，TM-5a和TM-5b不能彼此异源二聚化。讨论了这些结果在理解TM多样性在细胞骨架动力学中的作用方面的意义。
