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Combining protein complementation and resonance energy transfer assays to determine if three different proteins are part of the same G protein signaling complex in living cells
Author(s) -
Rebois Robert Victor,
Dupre Denis J.,
Robitaille Melanie,
Hebert Terence E.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb4-b
Subject(s) - bimolecular fluorescence complementation , heterotrimeric g protein , förster resonance energy transfer , yellow fluorescent protein , green fluorescent protein , protein–protein interaction , g protein , microbiology and biotechnology , complementation , protein subunit , fluorescence , chemistry , signal transduction , biophysics , biology , biochemistry , mutant , gene , physics , quantum mechanics
The N‐ and C‐terminal fragments of yellow fluorescent protein (YN and YC) expressed separately or together are not fluorescent. However, if YN and YC are brought together by fusing them to proteins that associate, a fluorescent protein can be reconstituted in a process called bimolecular fluorescence complementation (BiFC). If this fluorescent protein is close to (<10 nm) a luciferase (Luc) tagged protein, bioluminescence resonance energy transfer (BRET) occurs when the energy produced by Luc is transferred to the fluorescent protein in a process we call BiFC/BRET. G protein‐mediated signal transduction involves the interaction of a receptor, a heterotrimeric G protein and an effector. BiFC/BRET experiments were used to determine if three separate G protein signaling components are part of the same complex in vivo . BiFC occurred when various G protein β and γ subunits tagged, respectively, with YN and YC were co‐expressed and the BiFC was plasma membrane localized. BiFC was detected between tagged Gβ 1–4 and Gγ 2 , Gγ 3 , Gγ 7 and Gγ 11 . YN‐Gβ 1 and YC‐Gγ 2 subunits together acted as an acceptor in BiFC/BRET experiments when co‐expressed with either adenylyl cyclase or the Kir3.1 K + channel subunit tagged with Luc. BiFC/BRET was observed in the absence of a receptor agonist and these interactions were sensitive to receptor stimulation. These results demonstrate the feasibility of using BiFC to identify novel interacting proteins for Gβγ subunits, and of combining BiFC and BRET to demonstrate the simultaneous presence of three signaling proteins in a complex.