CLUSTERING OF GPI-ANCHORED FOLATE RECEPTOR-INDEPENDENT OF BOTH CROSS-LINKING AND ASSOCIATION WITH CAVEOLIN

The distribution of the glycosyl-phosphatidylinositol (GPI)-anchored f olate receptor (FR) in a diffuse pattern vs. functional clusters assoc iated with caveolae has been debated. The equivocal nature of direct l ocalization studies is due to possible experimental artifacts such as cross-linking of the protein by the antibody probes prior to fixation and alternatively the use of a disruptive fixation method. Such studie s have also been complicated by the use of cells that vastly overexpre ss FR. In this study a monovalent probe, i.e., a biotinylated folate a ffinity analogue was used to covalently label FR. Cells expressing mod erate levels of FR, i.e., JAR epithelial cells expressing FR-alpha and recombinant CHO fibroblasts expressing FR-beta, were used. The affini ty label and either caveolin or antigenic sites on FR were localized b y electron microscopy using colloidal gold conjugated antibody probes post-embedding in the relatively permeable LR White resin. The method avoided both receptor cross-linking and early fixation steps and also enabled the use of transport permissive conditions while labeling FR a t the cell surface. The results indicate that in steady-state FR is no t significantly colocalized with caveolin. However, the receptor molec ules occur predominantly in clusters, independent of cross-linking, pr oviding a physical basis for the observed kinetics of receptor interna lization and recycling during folate transport. Evidence is also prese nted to suggest that early mild fixation will disrupt the clustering o f FR.