Alternatively spliced exons of the β tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon

The rat β-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle β-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle β-TM. Skeletal muscle β-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal β gene products, fibroblast TM-1 and β skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 μM for β Sk TM, 8.5 for TM-6/10, 25 μM for TM-1, and 30 μM for TM-7/11 at an F-actin concentration of 42 μM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the β skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the α gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed β gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other β gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The β tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.