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Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads
Ecole Polytechnique Fédérale de Lausanne, Switzerland.ORCID-id: 0000-0002-4762-4887
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2011 (Engelska)Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, nr 42Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2011. Vol. 133, nr 42
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Biofysik Biokemi och molekylärbiologi Analytisk kemi
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URN: urn:nbn:se:kth:diva-178093DOI: 10.1021/ja205302gISI: 000296678200034PubMedID: 21910424Scopus ID: 2-s2.0-80055011129OAI: oai:DiVA.org:kth-178093DiVA, id: diva2:877630
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Tillgänglig från: 2015-12-07 Skapad: 2015-12-07 Senast uppdaterad: 2017-12-01Bibliografiskt granskad

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Piguet, Joachim

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Journal of the American Chemical Society
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