LOW-MOLECULAR-WEIGHT RAT FIBROBLAST TROPOMYOSIN-5 (TM-5) - CDNA CLONING, ACTIN-BINDING, LOCALIZATION, AND COILED-COIL INTERACTIONS

Previous studies have shown that three distinct genes encode six isofo rms of tropomyosin (TM) in rat fibroblasts: the a gene encodes TM-2, T M-3, TM-5a, and TM-5b, the beta gene encodes TM-1, and the TM-4 gene e ncodes TM-4. Here we report the characterization of a cDNA clone encod ing the most recent rat fibroblast TM to be identified, herein referre d 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 alp ha TM gene. The cDNA clone is approximately 1.7 kb and encodes a prote in of 248 amino acids. Using two-dimensional gel electrophoresis, the TM-5 protein was found to co-migrate with fibroblast TM-5a and 5b. Com parison of the amino acid sequences of TM-5 to other fibroblast isofor ms encoded by the alpha, beta, and TM-4 genes revealed a high degree o f homology, although there were regions of divergence among the differ ent isoforms. The gene encoding TM-5 is expressed in all tissues exami ned including skeletal muscle, stomach, heart, liver, kidney, uterus, spleen, brain, and diaphragm. However, Northern blot and RNase protect ion analyses revealed the presence of different mRNAs in fibroblasts, striated muscle (skeletal and diaphragm), and brain, which are express ed via alternative RNA splicing and the use of alternative promoters. The TM-5 protein was expressed in a bacterial system and tested for it s ability to bind actin in vitro and in vivo. The apparent TM associat ion constant (K-a) was taken as the free concentration at half saturat ion and was found to be 3 mu M for TM-5 compared to 2 mu M for TM-5b a t an F-actin concentration of 42 mu M. When fluorescently-labeled TM-5 was microinjected into living rat fibroblasts, it localized to the st ress fibers and ruffles of the leading lamella. The coiled-coil intera ctions of TM-5 with other low and high molecular weight TM isoforms we re studied. TM-5 and TM-4 were capable of dimerizing with each other a s 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 implic ations of these results in understanding the role of TM diversity in c ytoskeletal dynamics are discussed. (C) 1996 Wiley-Liss, Inc.