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- W2078267701 abstract "Oligomerization of membrane receptors in living cell membranes plays an important role in regulation of receptor activity and trafficking. Forster resonance energy transfer (FRET) techniques are often employed to detect receptor oligomerization using receptors fused with fluorescent/luminescent proteins. However, the large size of fluorescent proteins often interferes localization and function of target receptors. Furthermore, controlling the labeling ratio (donor/acceptor), which is important for analysis of FRET, is usually difficult when two fusion receptors with different colors are co-expressed. Posttranslational labeling methods using a short tag peptide and a fluorescent probe that specifically binds to the tag enable a smaller size of label and easy control of labeling ratio in multicolor labeling [1,2]. We recently developed a quick (< 1 min) and surface-specific tag-probe method using a high-affinity heterodimeric coiled-coil formation between the E3 tag (EIAALEK)3 attached to the target receptor and the Kn probes (KIAALKE)n (n = 3 or 4) labeled with a fluorophore [3]. Here we examine oligomerization of the metabotropic glutamate receptor (mGluR) using this novel technique. The receptors were labeled with Rhodamine Green (donor) or Tetramethylrhodamine (acceptor) fluorophores. A constant FRET signal was observed for mGluRs transiently expressed in CHO cells, indicating constitutive oligomer formation. Spectral imaging and demixing of emission spectra abolish crosstalk between channels that is inevitable in conventional filter detection therefore simplify quantification of FRET efficiency from sensitized acceptor emission, allowing analysis of stoichiometry of the oligomerization.[1] Murel et al. Nat. Mathods (2008) 5, 561-567.[2] Meyer et al. PNAS (2006) 103, 2138-2143.[3] Yano et al. ACS Chem. Biol. (2008) 3, 341-345" @default.
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- W2078267701 date "2010-01-01" @default.
- W2078267701 modified "2023-09-29" @default.
- W2078267701 title "Oligomerization of Membrane Receptors: FRET Analysis Using Coiled-Coil Tag-Probe Labeling and Spectral Imaging" @default.
- W2078267701 doi "https://doi.org/10.1016/j.bpj.2009.12.1569" @default.
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