Depletion of the lipid raft constituents, sphingomyelin and ganglioside, decreases serotonin binding at human 5-HT7(a) receptors in HeLa cells

Aim: The localization and function of several G protein‐coupled receptors, including β‐adrenergic receptors and NK 1 receptors, are regulated via lipid rafts in the plasma membrane. These domains are enriched in cholesterol, gangliosides and sphingolipids, and play an important role in regulating signal transduction in most cell types. Serotonin (5‐hydroxytryptamine, 5‐HT), acting via 14 different receptors, regulates as diverse effects as mood, metabolism and smooth muscle contraction. 5‐HT₇ receptors are involved in the regulation of depression, circadian rhythms, thermoregulation and vasodilatation. Ligand binding and signalling via the 5‐HT₇ receptor are regulated by membranous cholesterol. Here we investigated the role of sphingomyelin and gangliosides on binding of 5‐HT to 5‐HT₇ receptors to further examine the role of lipid raft constituents on 5‐HT₇ receptor function. Methods: HeLa cells stably transfected with the human 5‐HT₇ receptor were treated with Fumonisin B₁ or (±)‐threo‐1‐Phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) to reduce sphingomyelin or ganglioside levels, respectively. The effects of these treatments were investigated by the 3‐4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl‐tetrazolium bromide (MTT) viability assay, cholesterol analysis and ³H]5‐HT binding studies on intact cells. Results: Treatments with 20 μm Fumonisin B₁ for 24 h or with 10 μm PDMP for 48 h had no effects of total levels if 5‐HT₇ receptors, but caused significant decreases in maximum ³H]5‐HT binding to 5‐HT₇ receptors. The effects were cholesterol‐independent as levels of cholesterol remained unaffected by either treatment. Conclusion: These data demonstrate a role for sphingomyelin and gangliosides in regulating binding of ³H]5‐HT to 5‐HT₇ receptors. These observations further strengthen that actions of 5‐HT via 5‐HT₇ receptors are dependent upon lipid raft integrity.