title: Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.
abstract: G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β(1)-adrenergic receptor (β(1)AR), the β(2)-adrenergic receptor (β(2)AR), and the γ-aminobutyric acid (GABA(B)) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β(1)ARs (monomers/dimers) and highest for GABA(B) receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β(1)-/β(2)ARs were apparently freely diffusing on the cell surface, GABA(B) receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA(B) receptor complexes. These data provide a spatiotemporal characterization of β(1)-/β(2)ARs and GABA(B) receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.
authors.affiliation: Institute of Pharmacology and Toxicology and Rudolf Virchow Center, Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine, University of Würzburg, 97078 Würzburg, Germany. davide.calebiro@toxi.uni-wuerzburg.de
authors.forename: Davide
authors.initials: D
authors.lastname: Calebiro
authors.forename: Finn
authors.initials: F
authors.lastname: Rieken
authors.forename: Julia
authors.initials: J
authors.lastname: Wagner
authors.forename: Titiwat
authors.initials: T
authors.lastname: Sungkaworn
authors.forename: Ulrike
authors.initials: U
authors.lastname: Zabel
authors.forename: Alfio
authors.initials: A
authors.lastname: Borzi
authors.forename: Emanuele
authors.initials: E
authors.lastname: Cocucci
authors.forename: Alexander
authors.initials: A
authors.lastname: Zürn
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
mesh_terms: D000465:Algorithms; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic; D016466:CHO Cells; D002232:Carbocyanines; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008055:Lipids; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D056004:Molecular Dynamics Simulation; D046912:Multiprotein Complexes / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics; D011869:Radioligand Assay; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry*; D018079:Receptors, GABA / Q000737:chemistry*; D053778:Thiazolidines
chemical_list: C569686:Alexa Fluor 647; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002232:Carbocyanines; D008055:Lipids; C086724:Lipofectamine; D046912:Multiprotein Complexes; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D018079:Receptors, GABA; D053778:Thiazolidines; D000242:Cyclic AMP; C037067:latrunculin A