Esser V, Limbird L E, Brown M S, Goldstein J L, Russell D W
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1988 Sep 15;263(26):13282-90.
The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.
低密度脂蛋白（LDL）受体的配体结合结构域包含一段40个氨基酸的富含半胱氨酸序列的七个不完全重复序列。每个重复序列都含有成簇的负电荷，这些负电荷被假定为配体结合位点。该蛋白的相邻区域，即生长因子同源区域，包含三个富含半胱氨酸的重复序列（A - C），其序列与配体结合结构域中的不同。为了剖析这些不同的富含半胱氨酸重复序列对配体结合的贡献，我们使用寡核苷酸定向诱变来改变人LDL受体的可表达cDNA，然后通过转染将其导入猴COS细胞。我们测量了突变受体结合LDL（其含有该受体的单一蛋白配体apoB - 100）和β迁移极低密度脂蛋白（β-VLDL，其含有apoB - 100加上另一种配体（apoE）的多个拷贝）的能力。结果表明，重复序列1对于两种配体的结合都不是必需的。重复序列2加3以及重复序列6加7对于LDL的最大结合是必需的，但对于β-VLDL则不是。重复序列5对于两种配体的结合都是必需的。生长因子同源区域中的重复序列A对于LDL的结合是必需的，但对于β-VLDL则不是。重复序列B对于配体结合不是必需的。这些结果支持了一种LDL受体模型，其中各种重复序列在配体结合中起累加作用，每个重复序列对结合事件都有单独的贡献。
