title: Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads.
abstract: G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.
authors.affiliation: Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
authors.forename: Sophie
authors.initials: S
authors.lastname: Roizard
authors.forename: Christophe
authors.initials: C
authors.lastname: Danelon
authors.forename: Ghérici
authors.initials: G
authors.lastname: Hassaïne
authors.forename: Joachim
authors.initials: J
authors.lastname: Piguet
authors.forename: Katrin
authors.initials: K
authors.lastname: Schulze
authors.forename: Ruud
authors.initials: R
authors.lastname: Hovius
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
authors.forename: Horst
authors.initials: H
authors.lastname: Vogel
mesh_terms: D001667:Binding, Competitive; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D016062:Porosity; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D013499:Surface Properties
chemical_list: D055767:Immobilized Proteins; D008024:Ligands; D043562:Receptors, G-Protein-Coupled