Abstract
According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-beta-cyclodextrin (MbetaCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged mu-opioid receptors (HEK FLAG-mu) or delta-opioid receptors (HEK FLAG-delta) with MbetaCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MbetaCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing mu-opioid receptors. The effects of MbetaCD may be due to uncoupling of the mu receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-mu cells, MbetaCD treatment of HEK FLAG-delta cells had no effect on acute inhibition or sensitization of adenylyl cyclase by delta-opioid agonists. The differential responses of mu- and delta-opioid agonists to cholesterol depletion suggest that mu-opioid receptors are more dependent on cholesterol for efficient signaling than delta receptors and can be partly explained by localization of mu- but not delta-opioid receptors in cholesterol- and caveolin-enriched membrane domains.
Highlights
Membrane cholesterol can alter the function of integral proteins, such as G protein-coupled receptors, through cholesterolprotein interactions and by changes in membrane viscosity [1]
3 The abbreviations used are: GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; SNC80, (ϩ)-4-[(␣R)-␣-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; DPDPE, [D-Pen2,5]-enkephalin; DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin; DPN, diprenorphine; CTAP, D-Phe-[CysTyr-D-Trp-Arg-Thr-Pen]Thr-NH2; MCD, methyl--cyclodextrin; CTB, cholera toxin B subunit; AC, adenylyl cyclase; TfR, transferrin receptor; ANOVA, analysis of variance; HEK, human embryonic kidney; FLAG epitope (DYKDDDDK); HEK FLAG-␦, HEK cells stably expressing FLAG-␦ receptor; HEK FLAG-, HEK cells stably expressingFLAG-receptor;Dulbecco’s modified Eagle’s medium (DMEM),Dulbecco’smodifiedEagle’smedium; FBS, fetal bovine serum; MBS, MES-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid; PBS, phosphate-buffered saline
The objective of this study was to directly compare the role of membrane cholesterol in modulating acute and chronic - and ␦-opioid signaling in the same cell systems using identical methods, including the following: 1) depletion of cholesterol by the cholesterol-sequestering agent methyl--cyclodextrin (MCD); 2) separation of cholesterolenriched membranes by sucrose gradient ultracentrifugation; and 3) clustering of lipid raft patches in whole cells with cholera toxin B subunit
Summary
Bilayer, sphingolipids [2], which allows for tight and organized packing that can precipitate the formation of specialized domains within the plasma membrane [3] These domains have become an area of intense research interest and have been termed lipid or membrane rafts [4]. Many G protein-coupled receptors and signaling proteins have been found to prefer cholesterol-enriched domains leading to the hypothesis that these domains can organize signaling molecules in the membrane to enhance or inhibit specific signaling events [7]. This includes - [8, 9], ␦- [10, 11], and -opioid receptors [12].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
