Savonen R, Obermoeller L M, Trausch-Azar J S, Schwartz A L, Bu G
Departments of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1999 Sep 3;274(36):25877-82. doi: 10.1074/jbc.274.36.25877.
The 39-kDa receptor-associated protein (RAP) is a specialized antagonist that inhibits all known ligand interactions with receptors that belong to the low density lipoprotein (LDL) receptor gene family. Recent studies have demonstrated a role for RAP as a molecular chaperone for the LDL receptor-related protein during receptor folding and trafficking within the early secretory pathway. In the present study, we investigated a potential role for RAP as a chaperone for the very low density lipoprotein (VLDL) receptor, another member of the LDL receptor gene family. Using intracellular cross-linking techniques, we found that RAP is associated with newly synthesized VLDL receptor. In the absence of RAP co-expression, newly synthesized VLDL receptor exhibited slower trafficking along the early secretory pathway, most likely due to misfolding of the receptor. The role of RAP in the folding of the VLDL receptor was further studied using an anchor-free, soluble VLDL receptor. Metabolic pulse-chase labeling experiments showed that while only 3% of the soluble VLDL receptor was folded and secreted in the absence of RAP co-expression, over 50% of the soluble receptor was secreted in the presence of RAP co-expression. The functions of RAP in VLDL receptor folding and trafficking were mediated by its carboxyl-terminal repeat but not by the amino-terminal and central repeats. Using truncated VLDL receptor constructs, we identified the RAP-binding site within the first three ligand-binding repeats of the VLDL receptor. Thus, our present study demonstrates that RAP serves as a folding and trafficking chaperone for the VLDL receptor via interactions of its carboxyl-terminal repeat with the three amino-terminal ligand-binding repeats of the VLDL receptor.
39 kDa受体相关蛋白（RAP）是一种特殊的拮抗剂，可抑制所有已知配体与属于低密度脂蛋白（LDL）受体基因家族的受体之间的相互作用。最近的研究表明，RAP在早期分泌途径中受体折叠和运输过程中作为LDL受体相关蛋白的分子伴侣发挥作用。在本研究中，我们研究了RAP作为LDL受体基因家族的另一个成员——极低密度脂蛋白（VLDL）受体的伴侣的潜在作用。使用细胞内交联技术，我们发现RAP与新合成的VLDL受体相关。在没有RAP共表达的情况下，新合成的VLDL受体在早期分泌途径中的运输较慢，这很可能是由于受体错误折叠所致。使用无锚定的可溶性VLDL受体进一步研究了RAP在VLDL受体折叠中的作用。代谢脉冲追踪标记实验表明，在没有RAP共表达的情况下，只有3%的可溶性VLDL受体折叠并分泌，而在有RAP共表达的情况下，超过50%的可溶性受体被分泌。RAP在VLDL受体折叠和运输中的功能由其羧基末端重复序列介导，而不是由氨基末端和中央重复序列介导。使用截短的VLDL受体构建体，我们在VLDL受体的前三个配体结合重复序列中鉴定出RAP结合位点。因此，我们目前的研究表明，RAP通过其羧基末端重复序列与VLDL受体的三个氨基末端配体结合重复序列的相互作用，作为VLDL受体的折叠和运输伴侣发挥作用。
