Grimsley P G, Quinn K A, Chesterman C N, Owensby D A
Centre for Thrombosis and Vascular Research, University of New South Wales, Sydney, Australia.
Thromb Res. 1999 May 1;94(3):153-64. doi: 10.1016/s0049-3848(98)00208-4.
Low density lipoprotein receptor family members characteristically bind 39-kDa receptor associated protein (RAP). Soluble forms of these receptors have been described in humans including the 515/85-kDa dimeric receptor, low density lipoprotein receptor-related protein (LRP/alpha2MR), which is involved in multiple processes including lipoprotein and protease metabolism. Here we demonstrate evolutionary conservation in the generation of these soluble RAP-binding proteins of high molecular weight, by identifying their presence in mammalian, avian, and reptilian sera as well as in the circulating haemolymph of a mollusc. Sera extracted on immobilized RAP, produced bands at approximately 500 kDa in radiolabeled ligand blots by using the LRP/alpha2MR-specific ligand, Pseudomonas exotoxin A (PEA). These findings suggest that circulating RAP-binding proteins with high molecular weight in vertebrates share features of LRP/alpha2MR (LRP-like molecules). RAP-binding molecules in the mammalian serum extracts were further characterized as LRP/alpha2MR homologues in Western blots by using antibodies against the 515-kDa alpha-chain of LRP/alpha2MR. Western blots of mammalian serum extracts using two monoclonal antibodies recognizing the 85-kDa transmembrane beta-chain suggested that a portion of the beta-chain's ectodomain remains associated with the alpha-chain, but the beta-chain's intracellular carboxy terminus is absent. These results are consistent with evolutionary conservation in the generation, composition, and ligand-binding ability of soluble LRP-like receptors and suggest that their presence is a necessary aspect of the receptor's function.
低密度脂蛋白受体家族成员通常会结合39 kDa的受体相关蛋白（RAP）。在人类中已描述了这些受体的可溶性形式，包括515/85 kDa的二聚体受体，即低密度脂蛋白受体相关蛋白（LRP/α2MR），它参与多种过程，包括脂蛋白和蛋白酶代谢。在这里，我们通过鉴定其在哺乳动物、鸟类和爬行动物血清以及一种软体动物的循环血淋巴中的存在，证明了这些高分子量可溶性RAP结合蛋白生成过程中的进化保守性。用固定化RAP提取的血清，通过使用LRP/α2MR特异性配体铜绿假单胞菌外毒素A（PEA），在放射性标记配体印迹中产生了约500 kDa的条带。这些发现表明，脊椎动物中循环的高分子量RAP结合蛋白具有LRP/α2MR（LRP样分子）的特征。通过使用针对LRP/α2MR 515 kDaα链的抗体，在Western印迹中将哺乳动物血清提取物中的RAP结合分子进一步鉴定为LRP/α2MR同源物。使用两种识别85 kDa跨膜β链的单克隆抗体对哺乳动物血清提取物进行Western印迹分析表明，β链胞外结构域的一部分仍与α链相关，但β链的细胞内羧基末端缺失。这些结果与可溶性LRP样受体的生成、组成和配体结合能力的进化保守性一致，并表明它们的存在是受体功能的一个必要方面。
