Ihida K, Predescu D, Czekay R P, Palade G E
Division of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, California 92093-0602, USA.
J Cell Sci. 1999 Feb;112 ( Pt 3):285-95. doi: 10.1242/jcs.112.3.285.
In previous studies, we have localized the platelet activating factor receptor (PAF-R) in situ on the surface of the endothelium in a number of microvascular beds without providing information on its intracellular location. In the present study, we used human umbilical vein cells (HUVECs) as a model to immunolocalize PAF-R by light and electron microscopic procedures. We raised two different polyclonal antibodies against synthetic peptides of the C- and N-terminal of PAF-R and used them for immunolocalization studies. By immunofluorescence, we found that the anti-C-terminal antibody (CPAF-R) stains an extensive intracellular tubular network. By electron microscopy, using a preembedding staining procedure, we detected PAF-R on the surface of the plasmalemma in a staining pattern similar to that described on microvascular endothelia in situ, but at a considerably lower density. Immunogold labeling of thin frozen sections revealed the presence of PAF-R on the plasmalemma, and especially in an extensive network of tubular-vesicular elements and vesicles associated with it. No detectable amounts of PAF-R were found in the endoplasmic reticulum (ER) or in Golgi cisternae. Double immunofluorescence labeling with antibodies for compartment marker proteins and PAF-R revealed that PAF-R localizes in an endosomal compartment. Confocal microscopy showed that PAF-R colocalizes in this compartment together with the transferrin receptor (Tf-R) and the thrombin receptor (TH-R), but it also showed that the colocalization was partial rather than complete. These findings suggest that the endosomal network is either discontinuous or, conversely, that the proteins in its membrane do not have a fully randomized distribution.
在先前的研究中，我们已将血小板活化因子受体（PAF-R）原位定位在多个微血管床的内皮表面，但未提供其细胞内定位的信息。在本研究中，我们使用人脐静脉细胞（HUVECs）作为模型，通过光学和电子显微镜方法对PAF-R进行免疫定位。我们制备了两种针对PAF-R C末端和N末端合成肽的不同多克隆抗体，并将它们用于免疫定位研究。通过免疫荧光，我们发现抗C末端抗体（CPAF-R）可对广泛的细胞内管状网络进行染色。通过电子显微镜，采用包埋前染色程序，我们在质膜表面检测到PAF-R，其染色模式与原位微血管内皮上描述的相似，但密度要低得多。薄冰冻切片的免疫金标记显示质膜上存在PAF-R，尤其是在广泛的管状-囊泡元件网络及其相关的囊泡中。在内质网（ER）或高尔基池中未发现可检测量的PAF-R。用隔室标记蛋白和PAF-R的抗体进行双重免疫荧光标记显示，PAF-R定位于内体隔室。共聚焦显微镜显示，PAF-R与转铁蛋白受体（Tf-R）和凝血酶受体（TH-R）在这个隔室中共定位，但也显示这种共定位是部分而非完全的。这些发现表明，内体网络要么是不连续的，要么相反，其膜中的蛋白质没有完全随机分布。
