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

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.