Ahn A H, Kunkel L M
Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.
J Cell Biol. 1995 Feb;128(3):363-71. doi: 10.1083/jcb.128.3.363.
Dystrophin, the protein product of the Duchenne muscular dystrophy locus, is a protein of the membrane cytoskeleton that associates with a complex of integral and membrane-associated proteins. Of these, the 58-kD intracellular membrane-associated protein, syntrophin, was recently shown to consist of a family of three related but distinct genes. We expressed the cDNA of human beta 1-syntrophin and the COOH terminus of human dystrophin in reticulocyte lysates using an in vitro transcription/translation system. Using antibodies to dystrophin we immunoprecipitated these two interacting proteins in a variety of salt and detergent conditions. We demonstrate that the 53 amino acids encoded on exon 74 of dystrophin, an alternatively spliced exon, are necessary and sufficient for interaction with translated beta 1-syntrophin in our assay. On the basis of its alternative splicing, dystrophin may thus be present in two functionally distinct populations. In this recombinant expression system, the dystrophin relatives, human dystrophin related protein (DRP or utrophin) and the 87K postsynaptic protein from Torpedo electric organ, also bind to translated beta 1-syntrophin. We have found a COOH-terminal 37-kD fragment of beta 1-syntrophin sufficient to interact with translated dystrophin and its homologues, suggesting that the dystrophin binding site on beta 1-syntrophin occurs on a region that is conserved among the three syntrophin homologues.
肌营养不良蛋白是杜氏肌营养不良症基因座的蛋白质产物，是一种膜细胞骨架蛋白，与一组整合蛋白和膜相关蛋白结合。其中，58-kD的细胞内膜相关蛋白——肌萎缩蛋白，最近被证明由三个相关但不同的基因家族组成。我们使用体外转录/翻译系统，在网织红细胞裂解物中表达了人β1-肌萎缩蛋白的cDNA和人肌营养不良蛋白的COOH末端。使用针对肌营养不良蛋白的抗体，我们在各种盐和去污剂条件下免疫沉淀了这两种相互作用的蛋白。我们证明，在我们的检测中，肌营养不良蛋白第74外显子（一个可变剪接外显子）编码的53个氨基酸对于与翻译后的β1-肌萎缩蛋白相互作用是必要且充分的。基于其可变剪接，肌营养不良蛋白可能因此以两种功能不同的形式存在。在这个重组表达系统中，肌营养不良蛋白的亲属——人肌营养不良相关蛋白（DRP或抗肌萎缩蛋白）和来自电鳐电器官的87K突触后蛋白，也与翻译后的β1-肌萎缩蛋白结合。我们发现β1-肌萎缩蛋白的COOH末端37-kD片段足以与翻译后的肌营养不良蛋白及其同源物相互作用，这表明β1-肌萎缩蛋白上的肌营养不良蛋白结合位点位于三个肌萎缩蛋白同源物中保守的区域。
