GRK2 inhibits the interaction of the α2A-adrenoceptor with Gi in a phosphorylation-independent manner

Olga Prokopets, Moritz Buenemann, Cornelius Krasel. University of Marburg, School of Pharmacy, Dept of Pharmacology and Clinical Pharmacy, Karl-von-Frisch-Str. 1, 35043 Marburg, Germany

Introduction: Most G-protein-coupled receptors (GPCRs) undergo homologous desensitisation after agonist stimulation. This two-step process involves phosphorylation of the agonist-occupied receptor by G-protein-coupled receptor kinases (GRKs), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptor. We wanted to study homologous desensitisation of the α2A-adrenergic receptor (α2AAR) by investigating the effects of GRKs and arrestins on G-protein recruitment.

Methods: We measured FRET between YFP-tagged α2AAR and CFP-tagged Gβ in transiently transfected HEK293T cells which were stimulated twice with 10 µM norepinephrine, and evaluated the association kinetics.

Results: We expected that cotransfection of GRKs and arrestins would have little effect on the initial interaction kinetics of the α2AAR with Gi but would slow it down during the second stimulation. However, we found that cotransfection of GRK2 and arrestin3 already slowed down the initial interaction kinetics; the half-life of the receptor:G-protein interaction was 0.2 s (n = 10) in the absence of GRK2/arrestin3 but 2.3 s (n = 12) in the presence of GRK2+arrestin3 (one-way ANOVA with Dunn post-hoc test: p < 0.001). This effect was independent of arrestin3 (half-life of 1.8 s, n = 7, p>0.05). No arrestin-independent effect of GRK2 only was observed with the Gs-coupled β1-adrenergic receptor. The effect could also not be recapitulated using GRK5 or GRK6 which showed half-lifes of 0.4 s (n = 9 for GRK5, n = 13 for GRK6). In the presence of the GRK2 C-terminus, well known to bind Gβγ subunits, the half-life of Gβ recruitment to the α2AAR was slightly longer (0.6 s, n = 13) but still significantly faster than in the presence of full-length GRK2 (p < 0.05). Interestingly, GRK2 catalytic activity was not required for its effect on Gβ recruitment to the α2AAR as there was no significant difference between GRK2 and GRK2 K220R (p > 0.05).

Conclusions: GRK2 can inhibit the interaction between the α2AAR and Gi in a phosphorylation-independent manner. We are currently investigating whether this effect can be observed for other Gi-coupled GPCRs and which regions in GRK2 mediate the inhibition.