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

  • Original Research Paper
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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.

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Acknowledgments

This work was supported by an Arthritis Foundation Investigator Award (to R.G.W.) and National Institutes of Health grant HL-28207 (to A.D.S.). The authors would like to thank Dr. Andrea Kalinoski in the University of Toledo Advanced Microscopy and Imaging Center (AMIC) and the Flow Cytometry Core facility for technical assistance and use of the equipment.

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Authors and Affiliations

  1. Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, 3000 Arlington Ave., MS1021, Toledo, OH, 43614, USA

    Joshua A. Vieth, Daniel Glaser, Kaitlyn Stiles & Randall G. Worth

  2. Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA

    Moo-kyung Kim & Alan D. Schreiber

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  1. Joshua A. Vieth
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  2. Moo-kyung Kim
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  3. Daniel Glaser
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  4. Kaitlyn Stiles
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  5. Alan D. Schreiber
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  6. Randall G. Worth
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Correspondence to Randall G. Worth.

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Responsible editor: Liwu Li.

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Vieth, J.A., Kim, Mk., Glaser, D. et al. FcγRIIa requires lipid rafts, but not co-localization into rafts, for effector function. Inflamm. Res. 62, 37–43 (2013). https://doi.org/10.1007/s00011-012-0548-1

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  • DOI: https://doi.org/10.1007/s00011-012-0548-1

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