Activation of G-Protein-Couple Receptors in Cell-Derived Plasma Membranes Supported on Porous Beads

S Roizard, CJA Danelon, G Hassaine, J Piguet, K Schulze, R Hovius, R Tampe, H Vogel

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)16868-16874
Number of pages7
JournalJournal of the American Chemical Society
Volume133
Issue number42
Publication statusPublished - 2011

Keywords

  • CWTS JFIS >= 2.00

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