Lipid Raft Localization of Cell Surface E-Selectin Is Required for Ligation-Induced Activation of Phospholipase Cγ

Abstract

E-selectin, an endothelial cell surface adhesion receptor for leukocytes, also acts as a signaling receptor. Upon multivalent ligation, E-selectin transduces outside-in signals into the endothelium leading to changes in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase signaling pathway. In addition, following leukocyte engagement, E-selectin associates via its cytoplasmic domain with components of the actin cytoskeleton and undergoes alterations in phosphorylation state that result in changes in gene expression. In this study, we show that E-selectin is localized in cholesterol-rich lipid rafts at the cell surface, and that upon ligation E-selectin clusters and redistributes in the plasma membrane colocalizing with a fraction of caveolin-1-containing rafts. In addition, we demonstrate that leukocyte adhesion via E-selectin results in association with and activation of phospholipase Cgamma (PLCgamma). Moreover, we show that disruption of lipid rafts with the cholesterol-depleting drug methyl-beta-cyclodextrin disrupts the raft localization of E-selectin as well as the ligation-induced association of E-selectin with PLCgamma, and subsequent tyrosine phosphorylation of PLCgamma. In contrast, cholesterol depletion has no effect on E-selectin-dependent mitogen-activated protein kinase activation. Thus, these findings demonstrate that the presence of E-selectin in lipid rafts is necessary for its association with, and activation of, PLCgamma, and suggest that this subcellular localization of E-selectin is related to its signaling function(s) during leukocyte-endothelial interactions.

Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

“…These domains may propagate signals into endothelial cells that regulate later events in the inflammatory cascade. 27 Our results, in conjunction with previous studies, underscore the importance of particular cell surface presentations of selectins or their ligands during leukocyte rolling on vascular surfaces under flow. Unlike several other adhesion receptors, P-selectin and E-selectin undergo rapid internalization from the cell surface.…”

“…This observation differed from a previous report that virtually all E-selectin was in lipid rafts of HUVECs. 27 That report used a detergent-free procedure in which sonicated cell membranes were fractionated on a discontinuous sucrose gradient, as originally used to isolate caveolae. 47 In this procedure, nonraft components might float in more buoyant fractions if they associate with raft structures.…”

“…We considered that these domains might be lipid rafts in view of a report that E-selectin was largely distributed in rafts of HUVECs. 27 To address this possibility, we extracted transfected CHO cells and HUVECs in cold 1% Triton X-100 and fractionated the extracts by centrifugation in a discontinuous OptiPrep gradient.The most buoyant fractions at the top of the gradient are enriched in cholesterol-rich raft microdomains that resist disruption with Triton X-100, whereas the less buoyant fractions at the bottom of the gradient contain nonraft components. We analyzed the fractions by blotting with probes to E-selectin, to caveolin-1 and GM1 (a membrane protein and a ganglioside enriched in lipid rafts), to transferrin receptor and moesin (a membrane protein and a cytosolic protein excluded from rafts), and to flotillin-1 (a membrane protein found in both raft and nonraft domains; Figure 5A,B).…”

MUC1 Initiates a Calcium Signal after Ligation by Intercellular Adhesion Molecule-1

“…Our use of a panel of enzymespecific inhibitors suggested that this signal is mediated through the classic phospholipase C (PLC)-IP 3 pathway (24), and involves Src, which may bridge MUC1 with calcium signal mediators, as Src reportedly physically associates with MUC1 (5) and can be upstream of PI 3-kinase and PLC␥ (25)(26)(27). The data showing the dependence of this signal on lipid rafts is complementary, as cholesterol depletion has previously been shown to disrupt the function of Src (28) and PLC (29,30).…”

Implications of Early Structural-Functional Changes in the Endothelium for Vascular Disease

“…Recently, it was reported that in cultured endothelium, E-selectin is localized in membrane lipid rafts and that, on ligation, E-selectin clusters and redistributes in a caveolin-1-rich plasma membrane fraction. 6 The Weibel Palade bodies, considered initially as storage and secretory granules for von Willebrand factor (vWf), are now reported to deposit also P-selectin, IL-8, eotaxin-3, endothelin-1, and angiopoietin-2; the regulated exocytosis of these bioactive components play a role in inflammation, hemostasis, and vascular tone. 7 Furthermore, the ECs that constitute a mass of Ϸ1 kg spread throughout the body (estimated total area of Ϸ7000 m 2 Ϸ6ϫ10 13 cells) possess an innate heterogeneity, expressed by differences in their structure and function according to the vessel they are lining or tissues in which they reside.…”