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.

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