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Combining resonance energy transfer methods reveals a complex between the α 2A ‐adrenergic receptor, Gα i1 β 1 γ 2 , and GRK2
Author(s) -
Breton Billy,
Lagacé Monique,
Bouvier Michel
Publication year - 2010
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/fj.10.164061
Subject(s) - beta adrenergic receptor kinase , g protein coupled receptor kinase , g protein coupled receptor , receptor , förster resonance energy transfer , kinase , chemistry , microbiology and biotechnology , biophysics , 5 ht5a receptor , biology , biochemistry , physics , fluorescence , quantum mechanics
Traditionally, G‐protein‐coupled receptor (GPCR) interactions with their G proteins and regulatory proteins, GPCR kinases (GRKs) and ar‐restins, are described as sequential events involving rapid assemblies/disassemblies. To directly monitor the dynamics of these interactions in living cells, we combined two spectrally resolved bioluminescence and one fluorescence resonance energy transfer (RET) methods. The RET combination analysis revealed that stimulation of the α 2A ‐adrenergic receptor (α 2A AR) leads to the recruitment of GRK2 at a receptor still associated with the Gα i1 β 1 γ 2 complex. The interaction kinetics of GRKs with Gγ 2 (2.8±0.4 s) and α 2A AR (5.2±0.5 s) were similar to that of the receptor‐promoted change in RET between Gα i1 and Gγ 2 (5.2±1.2 s), and persisted until the translocation of βarrestin2 to the receptor, indicating that GRK2 remains associated to the receptor/G‐protein complex for longer periods than anticipated. Moreover, GRK2 or a kinase‐deficient GRK2 mutant, but not GRK5, potentiated the receptor‐promoted changes in RET between Gα i1 and Gγ 2 and abrogated the α 2A AR‐stimulated calcium response, suggesting that the recruitment of GRK2 to the complex contributes to the structural rearrangement and functional regulation of the signaling unit, independently of the kinase activity. RET combination analysis revealed unanticipated dynamics in GPCR signaling and will be applicable to many biological systems.—Breton, B., Lagace, M., Bouvier, M. Combining resonance energy transfer methods reveals a complex between the α 2A ‐adrenergic receptor, Gα i1 β 1 γ 2 , and GRK2. FASEBJ. 24, 4733–4743 (2010). www.fasebj.org