Involvement of lipid rafts in G protein-coupled monoamine receptor trafficking and signaling : A pharmacological approach

Sammanfattning: The present work focused on lipid raft-mediated modulation of signaling and trafficking of serotonin (5-HT) and dopamine receptors. The 5-HT system is one of the most complex neurotransmitter systems, with a wide distribution both in the CNS and in the periphery. It is involved in the regulation of many biological functions as diverse as mood, metabolism and cardiovascular tone. 5-HT acts via at least 14 receptors, divided into seven subgroups according to sequence homology and mode of signal transduction. All of them are G protein-coupled receptors (GPCRs), except for the 5-HT3 receptor, which is a ligand-gated ion channel. The dopamine system is involved in the motor, cognitive and reward systems of the brain. Dopamine acts via five receptors, all GPCRs, divided into D1-like and D2-like receptors according to their mode of signal transduction. Dysregulation of the dopamine system is the underlying cause of addiction, schizophrenia and Parkinson s disease (PD). The Singer-Nicholson fluidic mosaic model has for many years been the general model for the structure of biological membranes. In recent years this view has been revised with the discovery of liquid ordered microdomains, lipid rafts, enriched in cholesterol and sphingolipids. These microdomains play important roles in regulating signal transduction and trafficking events. A subgroup of lipid rafts, caveolae, is flask-shaped invaginations of the plasma membrane. They contain caveolin (Cav) proteins that can interact with other proteins, including GPCRs. Recently it was discovered that lipid rafts and caveolae participate in endocytosis and receptor trafficking. Although the exact mechanism for these processes is not yet fully understood, it seems clear that there can be alternative routes of receptor internalization than the classical endosomatic pathway via clathrin coated vesicles. In the present work we found that decreases of cholesterol, gangliosides, sphingomyelin or Cav-1 (by RNA interference) levels, attenuated maximum agonist and antagonist binding to 5-HT7 receptors. This could not be explained by a mere reduction in total receptor protein levels. Furthermore, signaling via 5-HT7 receptors was attenuated by cholesterol depletion with HMG-CoA reductase inhibitors, statins, which are widely used in the treatment of atherosclerosis. Reduction of cholesterol levels also attenuated signaling via 5-HT1A receptors in primary neuronal cultures. Furthermore, we could detect dopamine D1 receptors in low density regions in a sucrose gradient, suggesting lipid raft localization. This was further confirmed by the finding that reduction of cholesterol levels inhibited signaling via dopamine D1 receptors in mouse striatal slices. These results give further indications that lipid raft play important roles in regulating monoamine GPCRs in vivo. We have also found that Cav-1, a key protein in caveolae, regulates cell surface expression of 5-HT7 receptors. Biochemical and cellular assays showed that 5-HT7 receptors are located in low density regions in a sucrose gradient together with Cav-1, indicating lipid raft localization. Furthermore, it was found that 5-HT7 receptors undergo agonist induced internalization and that this could be inhibited by reducing Cav-1 expression with RNA interference. The internalization could also be inhibited with genistein, known to block lipid raft-mediated internalization. In contrast, hypertonic sucrose solution could not inhibit internalization. Altogether this suggests that 5-HT7 receptors are internalized through a clathrin-independent mechanism that is dependent on Cav-1.