The role of cholesterol and sphingolipids in the dopamine D receptor and G protein distribution in the plasma membrane.

G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein-lipid interactions play a pivotal role in cell signalling. Our previous results showed that although Gαs and Gαi prefer different types of membrane domains they are both co-localized with the D receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi and their heterotrimers, as well as the D receptor. We measured the lateral diffusion and membrane localization of investigated proteins using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by lifetime imaging microscopy (FLIM). The treatment with either methyl-β-cyclodextrin or Fumonisin B led to the disruption of cholesterol-sphingolipids containing domains and changed the diffusion of Gαi and the D receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D receptor exists in several different membrane localizations, depending on the receptor's conformation. We conclude that the inactive G protein heterotrimers are localized in the low-density membrane phase, from where they displace upon dissociation into the membrane-anchor- and subclass-specific lipid domain.