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Biophysical Journal
Article . 2013 . Peer-reviewed
License: Elsevier Non-Commercial
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Biophysical Journal
Article
License: Elsevier Non-Commercial
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Probing the Stoichiometry and Geometry of M3 Acetylcholine Receptors at the Plasma Membrane

Authors: Patowary, Suparna; Alvarez-Curto, Elisa; Xu, Tian Rui; Holz, Jessica D.; Oliver, Julie A.; Milligan, Graeme; Raicu, Valerică;

Probing the Stoichiometry and Geometry of M3 Acetylcholine Receptors at the Plasma Membrane

Abstract

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins in nature. GPCRs cascade signals from outer environment into cells and hence, are the target of more than 60% of modern clinical drugs. Determination of oligomerization of GPCRs is subject to significant controversy in the literature. We have investigated the quaternary structure of human muscarinic acetylcholine receptor type 3 (hM3) in living cells using Forster Resonance Energy Transfer (FRET) and two-photon excitation in an optical micro-spectroscopic set-up [Raicu et al, Nature Photonics, 2009]. The wild-type form of the hM3 receptor was fused to Cerulean while its mutated form, activated solely by a synthetic ligand (RASSL), was fused to Citrine and expressed constitutively in Flp-InTM T-RExTM 293 cells [Alvarez-Curto et al, Journal of Biological Chemistry, 2010]. When WT-hM3-Citrine and RASSL-hM3-Cerulean were co-expressed in same cells, excited donor (Cerulean), transferred its energy to nearby acceptors (Citrine) through FRET. Apparent FRET efficiency (Eapp) distribution maps were obtained for the imaged section of the cells by unmixing acquired spectrally resolved images using elementary donor and acceptor spectra. By selecting pixels in the Eapp image corresponding to the plasma membrane, a FRET efficiency histogram was obtained displaying the number of pixels in a particular range of Eapp values versus the corresponding Eapp value. The Eapp histograms were analyzed using a FRET theory [V. Raicu, Journal of Biological Physics, 2007], predicting various numbers and positions of peaks in the histograms for various sizes and geometry of oligomers. Eapp histograms of all cells were best fitted by a rhombus tetramer model. By also simulating the amplitudes of the peaks, we determined that hM3 receptors form both dimers and rhombus tetramers at the plasma membrane, and that their proportion remained largely unaffected by CNO binding.

Subjects by Vocabulary

Microsoft Academic Graph classification: Chemistry Cerulean Geometry Ligand (biochemistry) Transmembrane protein Crystallography Förster resonance energy transfer Tetramer Protein quaternary structure Acetylcholine receptor G protein-coupled receptor

Keywords

Biophysics

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citations
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
0
Average
Average
Average
hybrid
Funded by
NSF| Simulation Studies of Protein Dynamics in Membranes
Project
  • Funder: National Science Foundation (NSF)
  • Project Code: 0750175
  • Funding stream: Directorate for Mathematical & Physical Sciences | Division of Chemistry
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