Combining resonance energy transfer methods reveals a complex between the alpha2A-adrenergic receptor, Galphai1beta1gamma2, and GRK2.
Traditionally, G-protein-coupled receptor (GPCR) interactions with their G proteins and regulatory proteins, GPCR kinases (GRKs) and arrestins, 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
Billy
B
Department of Biochemistry, Institute for Research in Immunology and Cancer, and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec, Canada.
Lagacé
Monique
M
Bouvier
Michel
M
D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*
D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D018341:Receptors, Adrenergic, alpha-2; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go