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

Pittenger, M. F., Kistler, A., Helfman, D. M. (October 1995) Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon. J Cell Sci, 108 ( . pp. 3253-65. ISSN 0021-9533 (Print)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/7593286

Abstract

The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-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 beta gene products, fibroblast TM-1 and beta 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 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. 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 beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha 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 beta 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 beta 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 beta 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.

Item Type: Paper
Uncontrolled Keywords: Actins/ metabolism Alternative Splicing Animals Calmodulin-Binding Proteins/ metabolism Exons/genetics Fibroblasts/metabolism Muscle, Skeletal/ metabolism Rats Recombinant Proteins/genetics/metabolism Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Sequence Analysis Tropomyosin/ genetics/metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > actin
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > exons > exon splicing
organism description > animal > mammal > rodent > rat
organism description > animal > mammal > rodent > rat

organs, tissues, organelles, cell types and functions > tissues types and functions > smooth muscle
CSHL Authors:
Communities: CSHL labs
Depositing User: Jessica Koos
Date: October 1995
Date Deposited: 11 Aug 2014 18:27
Last Modified: 06 Nov 2017 20:10
Related URLs:
URI: http://repository.cshl.edu/id/eprint/30633

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