Differential Effect of Membrane Cholesterol Removal on μ- and δ-Opioid Receptors

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. -ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing -opioid receptors. The effects of M␤CD may be due to uncoupling of the 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-cells, M␤CD treatment of HEK FLAG-␦ cells had no effect on acute inhibition or sensitization of adenylyl cyclase by ␦-opioid agonists. The differential responses of -and ␦-opioid agonists to cholesterol depletion suggest that -opioid receptors are more dependent on cholesterol for efficient signaling than ␦ receptors and can be partly explained by localization of -but not ␦-opioid receptors in cholesterol-and caveolin-enriched membrane domains.

Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

“…We now report the distribution of AC isoform proteins in lipid and non-lipid rafts, using sucrose gradient fractionation samples from these cells. Among the AC isoforms only AC5/6 protein (using an antibody that recognizes both AC5 and AC6) was expressed mainly in lipid rafts in these kidney cells, in agreement with previous reports (13, 14, 16, 17, 23) (Figures 2A and 2B, upper panels ). In the basal state, more AC5/6 was distributed in lipid rafts than in non-lipid rafts and to a greater extent in HEK-hD 1 R cells (84.5±2.3 % of total density units, DU) than HEK-hD 5 R cells (69±3.1 % total DU) (P<0.05, n=4, ANOVA) (Figure 2A).…”

“…AC3 protein was also present in lipid and non-lipid rafts in HEK-hD 1 R, in agreement with previous reports (13, 14, 23), and hRPT cells, but present only non-lipid rafts in HEK-hD 5 R cells, supporting the aforementioned notion that the concomitant expression of a specific receptor may alter AC localization in membrane microdomains. Fenoldopam decreased AC3 protein in lipid rafts in HEK-hD 1 R and hRPT cells but had no effect in HEK-hD 5 R cells (Figure 2C).…”

“…βCD, a cholesterol-depleting reagent, has been used to determine the effect of disruption of lipid rafts on the distribution of proteins in cell membranes (16-20, 23). The cells were treated with βCD (2%) at 37°C for 1hr, and the lysates were subjected to sucrose gradient centrifugation as in previous experiments (12, 27) (Figure 3A).…”

“…The association of GPCRs with specific AC isoforms has been reported. AC5 and AC6 are associated with: β 2 adrenergic receptors (β 2 AR) in lipid rafts in cardiac, vascular, and bronchial smooth muscle cells (16-18); nicotinic acetylcholine receptor (nAChR) in lipid rafts in pheochromocytoma cells (PC12 cells) (19, 20); and μ- but not δ-opioid receptor in lipid rafts in human embryonic (HEK) cells (23). Toll-like receptor 4 is associated with AC6 in lipid rafts in a murine macrophage cell line (24), while stomatin-related olfactory protein and AC3 are found in lipid rafts in olfactory cilia (22).…”

Down-regulation of c-Cbl by Morphine Accounts for Persistent ERK1/2 Signaling in δ-Opioid Receptor-expressing HEK293 Cells

“…Representative blots of three different experiments are shown. data (58). Despite the failure of regulating DOR localization, our studies revealed that chronic morphine treatment results in the recruitment of the entire pool of membrane raft located c-Src to non-raft membranes.…”