Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human umbilical vein endothelial cells

In previous studies, we have localized the platelet activating factor recep tor (PAF-R) in situ on the surface of the endothelium in a number of microv ascular beds without providing information on its intracellular location. I n the present study, we used human umbilical vein cells (HUVECs) as a model to immunolocalize PAF-R by light and electron microscopic procedures. We r aised two different polyclonal antibodies against synthetic peptides of the C- and N-terminal of PAF-R and used them for immunolocalization studies. B y immunofluorescence, we found that the anti-C-terminal antibody (CPAF-R) s tains an extensive intracellular tubular network. By electron microscopy, u sing a preembedding staining procedure, we detected PAF-R on the surface of the plasmalemma in a staining pattern similar to that described on microva scular endothelia in situ, but at a considerably lower density. Immunogold labeling of thin frozen sections revealed the presence of PAF-R on the plas malemma, and especially in an extensive network of tubular-vesicular elemen ts and vesicles associated with it. No detectable amounts of PAF-R were fou nd in the endoplasmic reticulum (ER) or in Golgi cisternae. Double immunofl uorescence labeling with antibodies for compartment marker proteins and PAF -R revealed that PAF-R localizes in an endosomal compartment. Confocal micr oscopy showed that PAF-R colocalizes in this compartment together with the transferrin receptor (Tf-R) and the thrombin receptor (TH-R), but it also s howed that the colocalization was partial rather than complete. These findi ngs suggest that the endosomal network is either discontinuous or, converse ly, that the proteins in its membrane do not have a fully randomized distri bution.