Hemmings L, Kuhlman P A, Critchley D R
Department of Biochemistry, University of Leicester, United Kingdom.
J Cell Biol. 1992 Mar;116(6):1369-80. doi: 10.1083/jcb.116.6.1369.
To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.
为了确定鸡平滑肌α-辅肌动蛋白氨基末端结构域（第1至245位氨基酸残基）内的肌动蛋白结合位点，我们在猴Cos细胞中表达了一系列α-辅肌动蛋白缺失突变体。缺失第2至19位、217至242位以及196至242位氨基酸残基的突变型α-辅肌动蛋白仍保留定位于肌动蛋白丝及丝末端的能力，这表明肌动蛋白结合位点位于第20至195位氨基酸残基之间。当在Cos细胞中表达截短的α-辅肌动蛋白（第1至290位氨基酸残基）时，该蛋白仅定位于丝末端。缺乏第196至242位氨基酸残基的缺失突变体保留了这种活性，证实这些残基对于肌动蛋白结合并非必需。通过在大肠杆菌中表达野生型和突变型肌动蛋白结合结构域作为融合蛋白，进一步确定了α-辅肌动蛋白中的肌动蛋白结合位点。对这些蛋白体外结合F-肌动蛋白能力的分析表明，结合位点位于第108至189位氨基酸残基之间。利用体内和体外试验，我们还表明，在几种肌动蛋白结合蛋白α-辅肌动蛋白家族成员中保守的序列KTFT（鸡平滑肌蛋白中的第36至39位氨基酸残基）对于肌动蛋白结合并非必需。最后，我们证实肌营养不良蛋白的氨基末端结构域在功能和结构上与α-辅肌动蛋白中的结构域同源。因此，当在Cos细胞中表达时，包含肌营养不良蛋白氨基末端区域（第1至233位氨基酸残基）与α-辅肌动蛋白第244至888位氨基酸残基融合的嵌合蛋白定位于含肌动蛋白的结构。此外，大肠杆菌表达的包含肌营养不良蛋白第1至233位氨基酸残基的融合蛋白能够在体外结合F-肌动蛋白。
