On the role of Gβγ in receptor-GRK2 interactions

C Krasel, O. Prokopets, D. Zindel, V. Wolters, M. Bünemann. Institute of Pharmacology and Clinical Pharmacy, Philipps-University Marburg, Marburg, Germany

Introduction: Most G-protein-coupled receptors undergo homologous desensitization by the successive action of GRKs (which phosphorylate agonist-occupied receptors) and arrestins (which bind to phosphorylated, agonist-occupied receptors, thereby competing with heterotrimeric G-proteins). While investigating the effect of GRK2 and/or arrestin on the interaction of Gi-coupled receptors with Gβγ subunits, we found that Gβγ and GRK2 influence each other in a receptor-specific manner.

Methods: We measured FRET between two Gi-coupled receptors, the α2A-adrenergic receptor (α2AAR) or the A1 adenosine receptor (A1R) tagged with YFP, and Gβ or GRK2 tagged with CFP. We also measured FRET between YFP-tagged GRK2 and CFP-tagged Gβ in the presence of untagged receptors. All experiments were performed in transiently transfected HEK293T cells (at least 8 experiments in at least 3 independent transfections) which were stimulated with 10 µM noradrenaline or 10 µM adenosine, as appropriate. Time constants were determined by nonlinear curve fitting with GraphPad Prism. Results are shown as half-lives (t1/2).

Results: GRK2 slowed down the interaction of CFP-tagged Gβ with YFP-tagged α2AAR (t1/2 without GRK2 0.2-0.3 s; with GRK2 0.6-1.3 s; p<0.05) but not with YFP-tagged A1R (t1/2 in both cases 0.5 s). Arrestin did not show any additional effects. The GRK2 effects did not require GRK2 catalytic activity. Furthermore, GRK2 did not affect the interaction of the Gαi1 subunit with the α2AAR, and the GRK2(D110A) mutation, which reduces the affinity of GRK2 to Gαq, did not attenuate the effect of GRK2 on the α2AAR-Gβ interaction. Further analysis revealed that a GRK2(R587Q) mutation, which drastically reduced its affinity to Gβγ, also abolished the effect of GRK2 on the recruitment of Gβ to α2AAR (t1/2 without GRK2 0.3 s; with GRK2 0.6 s [p<0.05 vs. “without GRK2” and GRK2(R587Q)]; with GRK2(R587Q) 0.4 s). FRET measurements between receptors and GRK2 revealed a strong agonist-dependent interaction of GRK2 with both the α2AAR and the A1R which was almost completely abolished for the GRK2(R587Q) mutant for both receptors. To ensure that the GRK2 was targeted to the membrane a CAAX motif was attached to the C-terminus of the GRK2-CFP fusion protein. CAAX-tagged GRK2 was localized constitutively to the plasma membrane and associated with the receptor much more rapidly than wild-type GRK2. GRK2-CAAX and GRK2(R587Q)-CAAX showed virtually identical dissociation rates from the α2AAR upon washout of agonist (t1/2 16 s for both), but the off-rate at the A1R was about two-fold faster for GRK2(R587Q)-CAAX (t1/2 12 s)than for GRK2-CAAX (t1/2 28 s; p<0.05). This suggests that GRK2 interaction with the A1R is stabilized by Gβγ subunits whereas GRK2 interaction with the α2AAR is independent of Gβγ.

Conclusions: We show that the interaction of GRK2 with Gi-coupled receptors is facilitated by Gβγ-mediated translocation of GRK2 to the membrane. This translocation is the time-limiting step for kinase-receptor interaction. We also show that GRK2 affects kinetics of Gβγ recruitment to the α2AAR but not the A1R. Finally, the complex of GRK2 with the A1R but not with the α2AAR is stabilized by Gβγ. This suggests that receptor-specific mechanisms govern the interaction with GRK2 and Gβγ.