Clustering of GPI-anchored folate receptor independent of both cross-linking and association with caveolin.
The distribution of the glycosyl-phosphatidylinositol (GPI)-anchored folate receptor (FR) in a diffuse pattern vs. functional clusters associated with caveolae has been debated. The equivocal nature of direct localization 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 studies have also been complicated by the use of cells that vastly overexpress FR. In this study a monovalent probe, i.e., a biotinylated folate affinity analogue was used to covalently label FR. Cells expressing moderate levels of FR, i.e., JAR epithelial cells expressing FR-alpha and recombinant CHO fibroblasts expressing FR-beta, were used. The affinity label and either caveolin or antigenic sites on FR were localized by 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 at the cell surface. The results indicate that in steady-state FR is not significantly colocalized with caveolin. However, the receptor molecules occur predominantly in clusters, independent of cross-linking, providing a physical basis for the observed kinetics of receptor internalization and recycling during folate transport. Evidence is also presented to suggest that early mild fixation will disrupt the clustering of FR.
Wu
M
M
Department of Biochemistry and Molecular Biology, Medical College of Ohio, C.S. 1008, Toledo, OH 43699, USA.
Fan
J
J
Gunning
W
W
Ratnam
M
M
D000818:Animals; D000918:Antibody Specificity; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D003432:Cross-Linking Reagents; D005404:Fixatives; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
C495540:CAV1 protein, human; D002352:Carrier Proteins; D051242:Caveolin 1; D022461:Caveolins; D003432:Cross-Linking Reagents; D005404:Fixatives; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C075939:biotin-SS-folate; D001710:Biotin; D005492:Folic Acid