Obermoeller L M, Warshawsky I, Wardell M R, Bu G
Edward Mallinkrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 Apr 18;272(16):10761-8. doi: 10.1074/jbc.272.16.10761.
The 39-kDa receptor-associated protein (RAP) is a molecular chaperone for the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that binds multiple ligands. The primary function of RAP has been defined as promotion of the correct folding of LRP, and prevention of premature interaction of ligands with LRP within the early secretory pathway. Previous examination of the RAP sequence revealed an internal triplication. However, the functional implication of the triplicated repeats was unknown. In the current study using various RAP and LRP domain constructs, we found that the carboxyl-terminal repeat of RAP possesses high affinities to each of the three ligand-binding domains on LRP, whereas the amino-terminal and central repeats of RAP exhibit only low affinity to the second and the fourth ligand-binding domains of LRP, respectively. Using truncated soluble minireceptors of LRP, we identified five independent RAP-binding sites, two on each of the second and fourth, and one on the third ligand-binding domain of LRP. By coexpressing soluble LRP minireceptors and RAP repeat constructs, we found that only the carboxyl-terminal repeat of RAP was able to promote the folding and subsequent secretion of the soluble LRP minireceptors. However, when the ability of each RAP repeat to inhibit ligand interactions with LRP was examined, differential effects were observed for individual LRP ligands. Most striking, both the amino-terminal and central repeats, but not the carboxyl-terminal repeat, of RAP inhibited the interaction of alpha2-macroglobulin with LRP. These differential functions of the RAP repeats suggest that the roles of RAP in the folding of LRP and in the prevention of premature interaction of ligand with the receptor are independent.
39 kDa受体相关蛋白（RAP）是低密度脂蛋白受体相关蛋白（LRP）的分子伴侣，LRP是一种结合多种配体的大型内吞受体。RAP的主要功能被定义为促进LRP的正确折叠，并防止配体在早期分泌途径中与LRP过早相互作用。先前对RAP序列的研究揭示了一个内部三联体。然而，三联重复序列的功能意义尚不清楚。在当前使用各种RAP和LRP结构域构建体的研究中，我们发现RAP的羧基末端重复序列对LRP上的三个配体结合结构域中的每一个都具有高亲和力，而RAP的氨基末端和中央重复序列分别仅对LRP的第二个和第四个配体结合结构域表现出低亲和力。使用LRP的截短可溶性微型受体，我们鉴定出五个独立的RAP结合位点，在LRP的第二个和第四个配体结合结构域上各有两个，在第三个配体结合结构域上有一个。通过共表达可溶性LRP微型受体和RAP重复构建体，我们发现只有RAP的羧基末端重复序列能够促进可溶性LRP微型受体的折叠和随后的分泌。然而，当检测每个RAP重复序列抑制配体与LRP相互作用的能力时，观察到了针对单个LRP配体的不同效应。最显著的是，RAP的氨基末端和中央重复序列而非羧基末端重复序列抑制了α2-巨球蛋白与LRP的相互作用。RAP重复序列的这些不同功能表明，RAP在LRP折叠以及防止配体与受体过早相互作用中的作用是独立的。
