title: The role of cholesterol and sphingolipids in the dopamine D1receptor and G protein distribution in the plasma membrane.
abstract: 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 Gαi3prefer different types of membrane domains they are both co-localized with the D1receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi3and their heterotrimers, as well as the D1receptor. 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 B1led to the disruption of cholesterol-sphingolipids containing domains and changed the diffusion of Gαi3and the D1receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi3prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D1receptor 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.
authors: Department of Physical Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
authors: Paweł
authors: P
authors: Mystek
authors: Department of Physical Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
authors: Przemysław
authors: P
authors: Dutka
authors: Department of Physical Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
authors: Magdalena
authors: M
authors: Tworzydło
authors: Department of Physical Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
authors: Marta
authors: M
authors: Dziedzicka-Wasylewska
authors: Department of Physical Biochemistry, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: a.polit@uj.edu.pl.
authors: Agnieszka
authors: A
authors: Polit
mesh_terms: D022461:Caveolins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D019204:GTP-Binding Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D021122:Protein Subunits / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000378:metabolism*; D013107:Sphingolipids / Q000378:metabolism*
chemical_list: D022461:Caveolins; D021122:Protein Subunits; D017447:Receptors, Dopamine D1; D013107:Sphingolipids; D002784:Cholesterol; D019204:GTP-Binding Proteins
keywords: Cholesterol; Dopamine D(1) receptor; G proteins; Lipids binding motifs; Sphingolipids