Original Research Paper

Inflammation Research

, Volume 62, Issue 1, pp 37-43

FcγRIIa requires lipid rafts, but not co-localization into rafts, for effector function

  • Joshua A. ViethAffiliated withDepartment of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences
  • , Moo-kyung KimAffiliated withDepartment of Medicine, University of Pennsylvania Perelman School of Medicine
  • , Daniel GlaserAffiliated withDepartment of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences
  • , Kaitlyn StilesAffiliated withDepartment of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences
  • , Alan D. SchreiberAffiliated withDepartment of Medicine, University of Pennsylvania Perelman School of Medicine
  • , Randall G. WorthAffiliated withDepartment of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences Email author 

Rent the article at a discount

Rent now

* Final gross prices may vary according to local VAT.

Get Access

Abstract

Objective

To determine if receptor localization into lipid rafts, or the lipid rafts themselves, are important for FcγRIIa effector functions.

Material

Wild-type FcγRIIa or mutant FcγRIIa(C208A) that does not translocate to lipid rafts were transfected into Chinese hamster ovary (CHO) cells which have been shown to be reliable cells for studying FcγR function.

Treatment

Cells were treated with buffer or methyl-β-cyclodextrin (MβCD) to deplete cholesterol and dissolve the structure of lipid rafts.

Methods

To evaluate lipid raft association, transfected CHO cells were lysed and centrifuged over a sucrose gradient. Fractions were run on SDS-PAGE and blotted for FcγRIIa or sphingolipid GM1 to illustrate the lipid raft fractions. Lateral mobility of GFP-tagged wild-type or mutant FcγRIIa was assessed using fluorescence recovery after photobleaching (FRAP) microscopy. Internalization of IgG-opsonized erythrocytes was assessed by fluorescence microscopy and uptake of heat-aggregated IgG (haIgG) was measured using flow cytometry.

Results

We observed that FcγRIIa(C208A) did not localize into lipid rafts. However, the mutant FcγRIIa retained lateral mobility and effector function similar to wild-type FcγRIIa. However, mutant FcγRIIa function was abolished upon treatment with MβCD.

Conclusions

Lipid rafts provide an essential component required for effector activities independent of receptor localization.

Keywords

Lipid raft Endocytosis Phagocytosis Fc receptors