Otey C A, Vasquez G B, Burridge K, Erickson B W
Department of Cell Biology, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1993 Oct 5;268(28):21193-7.
The actin cross-linking protein alpha-actinin binds to the cytoplasmic domain of the beta 1 subunit of integrin, suggesting that alpha-actinin may form a direct link between the actin cytoskeleton and the transmembrane fibronectin receptor. In this study, we have used short synthetic peptides to localize the binding site for alpha-actinin within the cytoplasmic domain of beta 1 integrin. Four 13-residue peptides were tested in both an affinity chromatographic assay and a solid-phase binding assay. The results indicated that two regions of sequence contribute to the binding of alpha-actinin: one near where the beta 1 cytoplasmic tail emerges from the membrane and a second segment located near the C terminus of the cytoplasmic tail. This binding pattern was investigated in more detail using an adaptation of the mimotope assay, in which each of the 32 overlapping sequential decapeptide segments from the beta 1 cytoplasmic domain was assembled on the head of a different plastic pin. The peptide-pin constructs were used to detect the binding of 125I-alpha-actinin. As predicted from our initial results, alpha-actinin was found to bind to two distinct clusters of peptide segments. This represents a novel use of the mimotope pin assay to map interactive sites on structural proteins.
肌动蛋白交联蛋白α-辅肌动蛋白可与整合素β1亚基的胞质结构域结合，这表明α-辅肌动蛋白可能在肌动蛋白细胞骨架与跨膜纤连蛋白受体之间形成直接连接。在本研究中，我们使用短合成肽来定位α-辅肌动蛋白在β1整合素胞质结构域内的结合位点。在亲和色谱分析和固相结合分析中测试了四种13个残基的肽。结果表明，两个序列区域有助于α-辅肌动蛋白的结合：一个靠近β1胞质尾巴从膜中伸出的位置，另一个片段位于胞质尾巴的C末端附近。使用模拟表位分析的改进方法更详细地研究了这种结合模式，其中来自β1胞质结构域的32个重叠连续十肽片段中的每一个都组装在不同塑料针的头部。肽-针构建体用于检测125I-α-辅肌动蛋白的结合。正如我们初步结果所预测的，发现α-辅肌动蛋白与两个不同的肽段簇结合。这代表了模拟表位针分析在绘制结构蛋白上的相互作用位点方面的新应用。
