Bu G, Maksymovitch E A, Geuze H, Schwartz A L
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1994 Nov 25;269(47):29874-82.
The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor that binds and endocytoses several structurally and functionally distinct ligands. Several of the ligands for LRP participate in both normal physiology and pathophysiology of the central nervous system. To begin to gain insights into the role of LRP in the central nervous system, we have analyzed the expression, subcellular distribution, and endocytic function of LRP in human glioblastoma U87 cells. These cells express an abundance of LRP at both the mRNA and protein levels. A 39-kDa protein, which copurifies with LRP and regulates its ligand binding activity, is also highly expressed in U87 cells. The subcellular localization of LRP and the 39-kDa protein was analyzed using scanning laser confocal and electron microscopy combined with immunolabeled U87 cells. At the plasma membrane, LRP was largely confined to clathrin-coated pits. Within cells, LRP and the 39-kDa protein partially colocalized within rough endoplasmic reticulum and the Golgi complex, suggesting a potential intracellular interaction between the two proteins. Little 39-kDa protein was found in endosomes in which LRP occurred abundantly. In examining the functional role of LRP in U87 cells, we found that LRP at the cell surface and along the cellular processes was functional in the binding and endocytosis of its ligands, and its activity therein was regulated by the 39-kDa protein. Using truncated recombinant 39-kDa protein constructs, we also demonstrated that distinct regions of the 39-kDa protein were responsible for inhibiting the binding of different LRP ligands on U87 cells. Our results thus strongly suggest several potential roles for LRP in brain protein and lipoprotein metabolism, as well as control of extracellular protease activity.
低密度脂蛋白受体相关蛋白（LRP）是一种多功能细胞表面受体，它能结合并内吞几种结构和功能不同的配体。LRP的几种配体参与中枢神经系统的正常生理和病理生理过程。为了深入了解LRP在中枢神经系统中的作用，我们分析了LRP在人胶质母细胞瘤U87细胞中的表达、亚细胞分布和内吞功能。这些细胞在mRNA和蛋白质水平上均大量表达LRP。一种与LRP共纯化并调节其配体结合活性的39 kDa蛋白在U87细胞中也高度表达。使用扫描激光共聚焦显微镜和电子显微镜结合免疫标记的U87细胞分析了LRP和39 kDa蛋白的亚细胞定位。在质膜上，LRP主要局限于网格蛋白包被小窝。在细胞内，LRP和39 kDa蛋白在粗面内质网和高尔基体复合体中部分共定位，提示这两种蛋白之间可能存在细胞内相互作用。在富含LRP的内体中几乎未发现39 kDa蛋白。在研究LRP在U87细胞中的功能作用时，我们发现细胞表面和细胞突起上的LRP在其配体的结合和内吞过程中起作用，并且其活性受39 kDa蛋白调节。使用截短的重组39 kDa蛋白构建体，我们还证明了39 kDa蛋白的不同区域负责抑制U87细胞上不同LRP配体的结合。因此，我们的结果强烈提示LRP在脑蛋白和脂蛋白代谢以及细胞外蛋白酶活性控制中具有几种潜在作用。
