Combining resonance energy transfer methods reveals a complex between the  2A-adrenergic receptor, G i1 1 2, 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 alpha(2A)-adrenergic receptor (alpha(2A)AR) leads to the recruitment of GRK2 at a receptor still associated with the G alpha(i1)beta(1)gamma(2) complex. The interaction kinetics of GRKs with G gamma(2) (2.8 +/- 0.4 s) and alpha(2A)AR (5.2 +/- 0.5 s) were similar to that of the receptor-promoted change in RET between G alpha(i1) and G gamma(2) (5.2 +/- 1.2 s), and persisted until the translocation of beta arrestin2 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 G alpha(i1) and G gamma(2) and abrogated the alpha(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 alpha(2A)-adrenergic receptor, G alpha(i1)beta(1)gamma(2), and GRK2. FASEB J. 24, 4733-4743 (2010). www.fasebj.org