Babcock G G, Fowler V M
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1994 Nov 4;269(44):27510-8.
Tropomodulin is a tropomyosin-binding protein that localizes to the pointed end of striated muscle thin filaments and caps the pointed end of tropomyosin-actin filaments in vitro. Results from previous studies have suggested that tropomyosin-tropomodulin interactions are isoform-specific. To investigate the molecular basis for the isoform specific interactions of tropomyosin and tropomodulin, we isolated a cDNA for chicken skeletal muscle tropomodulin. The derived amino acid sequence of muscle tropomodulin is 86% identical with that of human erythrocyte tropomodulin, indicating that tropomodulins are highly conserved proteins. Multiple mRNAs seen on Northern blots of chicken skeletal muscle mRNA can be accounted for by multiple polyadenylation signals in the 3'-untranslated region of the cDNA. 125I-Labeled skeletal muscle and erythrocyte tropomyosins were assayed for binding bacterially expressed muscle tropomodulin using a solid phase binding assay. Unexpectedly, skeletal muscle and erythrocyte tropomyosins bound with similar affinities to muscle tropomodulin (Kd values approximately 0.2 microM). However, cross-competition studies using erythrocyte and skeletal muscle tropomyosins indicated that they bound different sites on tropomodulin. Competition studies using recombinant fragments of tropomodulin to map the binding domains for the two tropomyosins demonstrated that residues 6-94 contained the skeletal muscle tropomyosin binding site and residues 90-184 contained the erythrocyte tropomyosin binding site. Binding of different regions of tropomodulin to muscle and non-muscle tropomyosins may permit interaction of tropomodulin with tissue-specific components or may influence the pointed end capping activity of tropomodulin.
原肌球蛋白调节蛋白是一种与原肌球蛋白结合的蛋白质，定位于横纹肌细肌丝的尖端，并在体外封闭原肌球蛋白 - 肌动蛋白丝的尖端。先前的研究结果表明，原肌球蛋白 - 原肌球蛋白调节蛋白的相互作用具有亚型特异性。为了研究原肌球蛋白和原肌球蛋白调节蛋白亚型特异性相互作用的分子基础，我们分离了鸡骨骼肌原肌球蛋白调节蛋白的cDNA。推导得到的肌肉原肌球蛋白调节蛋白的氨基酸序列与人类红细胞原肌球蛋白调节蛋白的氨基酸序列有86%的同一性，这表明原肌球蛋白调节蛋白是高度保守的蛋白质。鸡骨骼肌mRNA的Northern印迹上出现的多个mRNA可以由cDNA 3'非翻译区中的多个聚腺苷酸化信号来解释。使用固相结合试验检测了125I标记的骨骼肌和红细胞原肌球蛋白与细菌表达的肌肉原肌球蛋白调节蛋白的结合情况。出乎意料的是，骨骼肌和红细胞原肌球蛋白与肌肉原肌球蛋白调节蛋白的结合亲和力相似（解离常数Kd值约为0.2 microM）。然而，使用红细胞和骨骼肌原肌球蛋白的交叉竞争研究表明，它们结合在原肌球蛋白调节蛋白的不同位点上。使用原肌球蛋白调节蛋白的重组片段进行竞争研究以绘制两种原肌球蛋白的结合域，结果表明第6 - 94位残基包含骨骼肌原肌球蛋白结合位点，第90 - 184位残基包含红细胞原肌球蛋白结合位点。原肌球蛋白调节蛋白的不同区域与肌肉和非肌肉原肌球蛋白的结合可能允许原肌球蛋白调节蛋白与组织特异性成分相互作用，或者可能影响原肌球蛋白调节蛋白的尖端封闭活性。
