Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.
Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.
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发表时间：
1989-12
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通讯作者：
D. Russell;Michael S. Brown;Joseph L. Goldstein
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作者：
D. Russell;Michael S. Brown;Joseph L. Goldstein
富含40个氨基酸的半胱氨酸序列的七个不完全重复序列构成低密度脂蛋白（LDL）受体的配体结合结构域。为了评估每个重复序列的贡献，在每个重复序列中单独进行三个定点突变：1）重复序列的缺失，2）保守的异亮氨酸被天冬氨酸取代，和3）保守的天冬氨酸被酪氨酸取代。将含有这些突变的cDNA转染到猿猴COS细胞中，并测定它们结合LDL（其含有500-kDa蛋白质配体（apoB-100））和β-迁移极低密度脂蛋白（β-VLDL）（其含有33-kDa配体（apoE）的多个拷贝）的能力。结果表明，两种配体的结合需要不同的重复组合。LDL结合需要重复3-7;这些重复中的任何一个的缺失显著降低LDL结合。相比之下，β-迁移极低密度脂蛋白的结合是不敏感的任何单一重复的损失与重复5，其损失减少了60%的结合的重要例外。当每个重复序列被两个取代突变中的任一个改变时，获得相同的效果。目前的研究结果表明，多个富含半胱氨酸的重复序列可能允许单个蛋白质通过采用不同的重复序列组合来结合几种不同的蛋白质配体。
Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.