Cholesterol Depletion Delocalizes Phosphatidylinositol Bisphosphate and Inhibits Hormone-stimulated Phosphatidylinositol Turnover

Linda J Pike,Joanne M Miller

doi:10.1074/jbc.273.35.22298

Abstract

Caveolae and detergent-insoluble, glycosphingolipid-enriched domains (DIGs) are cholesterol-enriched membrane domains that have been implicated in signal transduction because a variety of signaling proteins as well as phosphatidylinositol bisphosphate (PtdInsP2) are compartmentalized in these domains. We report here that depletion of cellular cholesterol leads to the inhibition of epidermal growth factor- and bradykinin-stimulated PtdIns turnover in A431 cells. This is associated with the loss of compartmentalization of epidermal growth factor receptors, Gq, and PtdInsP2 in the low density membrane domains. Replacement of cellular cholesterol leads to the reorganization of signaling molecules in the low density domains and the reestablishment of hormone-stimulated PtdIns hydrolysis. Oxysterol derivatives show a variable ability to functionally replace the cholesterol in this system. These data are consistent with the hypothesis that localization of signaling proteins and lipids to cholesterol-enriched domains is required for the proper function of hormone-stimulated PtdIns turnover.

Highlights

Caveolae are small, plasma membrane invaginations first identified by Palade and co-workers in the 1950s [1]
Because caveolae/DIGs are membrane domains that are enriched in cholesterol, we wondered what effect cholesterol depletion would have on the integrity of these domains, their ability to compartmentalize PtdInsP2, and the process of hormone-stimulated PtdIns turnover
The cholesterol dependence of hormone-stimulated PtdIns hydrolysis may be caused by the localization of this signaling pathway to cholesterol-enriched membrane domains

Summary

EXPERIMENTAL PROCEDURES

Materials—Anti-caveolin-1 and anti-caveolin-2 antibodies were from Transduction Laboratories. The polyclonal antibody to the EGF receptor (DB-1) was prepared as described previously [25]. Isolation of Caveolin-enriched Membranes—One 150-mm plate of A431 cells was washed in phosphate-buffered saline and scraped into 1 ml of ice-cold 150 mM Na2CO3, pH 11, 2 mM EDTA. Lipid Analyses—Aliquots (800 ␮l) of each sucrose density gradient fraction were extracted in chloroform: methanol:HCl and inositol phospholipids analyzed as described previously [22]. Cells were switched to inositol-free DMEM containing 1 mg/ml bovine serum albumin. The sterol was added in small aliquots and the solution stirred until clear. This yields a solution that contains 6.8 mM sterol. Complexes were diluted into inositol-free DMEM containing 1 mg/ml bovine serum albumin to a final concentration of 0.2 mM

RESULTS

Cholesterol Depletion Inhibits PI Turnover

Control ϩCyclodextrin ϩBovine serum albumin

DISCUSSION