Czekay R P, Orlando R A, Woodward L, Adamson E D, Farquhar M G
Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla 92093, USA.
J Cell Sci. 1995 Apr;108 ( Pt 4):1433-41. doi: 10.1242/jcs.108.4.1433.
The receptor-associated protein, RAP, is a chaperonin-like molecule that binds to two members of the low density lipoprotein receptor (LDLR) superfamily-megalin (gp330) and the LDL receptor-related protein (LRP). In F9 embryonal carcinoma cells, expression of RAP mRNA increases when differentiation is induced with retinoic acid and dibutyryl-cyclic AMP. We have investigated the expression of megalin and LRP and their interaction with RAP in F9 cells using biochemical and immunocytochemical methods. Both receptors are expressed in uninduced F9 cells, but only megalin co-precipitates with RAP. When F9 cells were induced to differentiate into parietal endoderm, the expression of megalin was dramatically increased. The expression of megalin exceeded that of LRP and RAP by an order of magnitude and both receptors co-precipitated with RAP. By immunoelectron microscopy, megalin and LRP were localized to clathrin-coated pits at the cell surface in both undifferentiated and differentiated F9 cells, whereas RAP was found mainly in the ER. A sizeable pool of LRP was also detected in the ER. When F9 cells were grown in suspension in the presence of RA and induced to develop into embryoid bodies, the expression of megalin and LRP segregated into different cell types: megalin was found in the outer epithelial layer and LRP in the stem cells of the inner core. Our results demonstrate that F9 cells induced to differentiate in monolayer culture express megalin, LRP and RAP, and RAP is capable of interacting simultaneously with both receptors. In embryoid bodies the expression of megalin and LRP diverges, and only megalin is expressed in the outer epithelial layer.(ABSTRACT TRUNCATED AT 250 WORDS)
受体相关蛋白（RAP）是一种伴侣蛋白样分子，它可与低密度脂蛋白受体（LDLR）超家族的两个成员——巨膜蛋白（gp330）和低密度脂蛋白受体相关蛋白（LRP）结合。在F9胚胎癌细胞中，用视黄酸和二丁酰环磷酸腺苷诱导分化时，RAP mRNA的表达会增加。我们运用生化和免疫细胞化学方法，研究了F9细胞中巨膜蛋白和LRP的表达及其与RAP的相互作用。两种受体均在未诱导的F9细胞中表达，但只有巨膜蛋白能与RAP共沉淀。当F9细胞被诱导分化为壁内胚层时，巨膜蛋白的表达显著增加。巨膜蛋白的表达比LRP和RAP高出一个数量级，且两种受体都能与RAP共沉淀。通过免疫电子显微镜观察，在未分化和分化的F9细胞中，巨膜蛋白和LRP都定位于细胞表面的网格蛋白包被小窝，而RAP主要存在于内质网中。在内质网中也检测到了大量的LRP。当F9细胞在视黄酸存在的情况下悬浮生长并被诱导发育成胚状体时，巨膜蛋白和LRP的表达分离到不同的细胞类型中：巨膜蛋白存在于外层上皮细胞层，LRP存在于内核的干细胞中。我们的结果表明，在单层培养中诱导分化的F9细胞表达巨膜蛋白、LRP和RAP，且RAP能够同时与两种受体相互作用。在胚状体中，巨膜蛋白和LRP的表达出现分化，只有巨膜蛋白在外层上皮细胞层中表达。（摘要截短至250字）
