Abstract

Receptor activation leads to the dynamic remodeling of the plasma membrane. Previous work using immunoelectron microscopy showed that aggregated high-affinity receptor for immunoglobulin E (FcRI) and aggregated Thy-1, a glycerophosphoinositol (GPI)-anchored protein, have distinct membrane distributions. We now report lipidomics analysis of FcRI- and Thy-1-enriched vesicles obtained by magnetic bead isolation in the absence of detergent. Protein analyses show that FcRI domains are enriched in receptors and associated signaling molecules, whereas Thy-1 domains are devoid of FcRI subunits. Positive and negative ion electrospray mass spectrometry demonstrated that both domains retained a complex mixture of phospholipid classes and molecular species, predominantly glycerophosphocholine, glycerophosphoethanolamine (GPE), and sphingomyelin as well as glycerophosphoserine and GPI lipids. Analysis of total acyl groups showed that < 50% of fatty acids in these domains are fully saturated, inconsistent with the recruitment of aggregated receptors or GPI-anchored proteins to liquid ordered domains. However, further analysis showed that FcRI domains contain two times more sphingomyelin and a high ratio of cholesterol to total fatty acid content compared with Thy 1-enriched domains. Remarkably, plasmenyl glycerophosphoethanolamine phospholipids (plasmalogen GPE) were also 2.5-3 times more abundant in FcRI domains than in the Thy-1 microdomains, whereas most diacyl GPE molecular species were equally abundant in the two domains.

Highlights

  • Receptor activation leads to the dynamic remodeling of the plasma membrane

  • Vesicles enriched in high-affinity receptor for immunoglobulin E (FceRI) or Thy-1 aggregates were immunoisolated on magnetic beads

  • The size and specificity of bound vesicles was determined by labeling immunoisolated FceRI vesicles with anti-IgE immune complexes conjugated to 10 nm colloidal gold, glutaraldehyde fixation, dehydration, and embedding in Epon

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Summary

Introduction

Receptor activation leads to the dynamic remodeling of the plasma membrane. Previous work using immunoelectron microscopy showed that aggregated high-affinity receptor for immunoglobulin E (FceRI) and aggregated Thy-1, a glycerophosphoinositol (GPI)-anchored protein, have distinct membrane distributions. Further analysis showed that FceRI domains contain two times more sphingomyelin and a high ratio of cholesterol to total fatty acid content compared with Thy 1-enriched domains. Receptor activation leads to the dynamic remodeling of the plasma membrane, facilitating the initiation, propagation, and termination of intracellular signaling. This remodeling of the membrane includes both the redistribution of membrane proteins and the activation of a host. These analyses have advanced the field, there are several limitations to lipid raft analysis by fractionation [15, 16]. This article is available online at http://www.jlr.org

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