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Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads.

Journal of the American Chemical Society (2011-09-14)
Sophie Roizard, Christophe Danelon, Ghérici Hassaïne, Joachim Piguet, Katrin Schulze, Ruud Hovius, Robert Tampé, Horst Vogel
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 647N NHS ester, BioReagent, suitable for fluorescence, ≥85% (coupling to amines)
Sigma-Aldrich
Atto 647, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Supelco
Atto 647N, BioReagent, suitable for fluorescence, ≥98.0% (HPCE)
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Atto 647N maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
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Atto 647 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
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Atto 633, BioReagent, suitable for fluorescence
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Atto 655, BioReagent, suitable for fluorescence, ≥85% (HPLC)
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Atto 655 NHS ester, BioReagent, suitable for fluorescence
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Atto 655-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Supelco
Atto 633 NHS ester, BioReagent, suitable for fluorescence, ≥90.0% (coupling to amines)
Sigma-Aldrich
Atto 647N-Biotin, BioReagent, ≥95% (HPCE), suitable for fluorescence
Sigma-Aldrich
Atto 647 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 647N azide
Sigma-Aldrich
Atto 655 amine, suitable for fluorescence
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Atto 633 amine, suitable for fluorescence
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Atto 633 iodoacetamide, suitable for fluorescence
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Atto 633 azide, suitable for fluorescence, ≥80.0% (HPCE)
Sigma-Aldrich
Atto 647N iodoacetamide
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Atto 647N amine
Sigma-Aldrich
Atto 655-maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling rate)

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