Gαq Contributes To The Recruitment Of GRK2 To M3-ACh Receptors

Valerie Wolters, Moritz Bünemann. Department of Pharmacology and Clinical Pharmacy, University Marburg, Marburg, Germany

G-protein-coupled receptor kinase 2 (GRK2) is well-known for its role in GPCR desensitisation. The GRK2 recruitment to the membrane is generally accepted to be mediated through Gβγ subunits. In addition an interaction between active Gαq subunits and GRK2 was observed (1). However, the function of this interaction remained elusive. Here we investigated the role of Gαq in membrane targeting of GRK2 and whether the Gαq-binding is important for the GPCR-GRK2 interaction. FRET microscopy (2) in single living HEK293T cells was performed to determine this interaction with high temporal resolution. Special attention was given to previously published mutants of GRK2 interfering with the interaction of GRK2 with Gαq (GRK2 (D110A) (3)) and Gβγ (GRK2 (R587Q) (4)) to investigate the influence of the different G-protein subunits. Data are shown as mean ± S.E.M. (n) and statistics has been performed by one way ANOVA with Bonferroni posthoc test.

An agonist-dependent development of FRET was observed between c-terminal fluorescently labelled GRK2 and M3-ACh receptor (Δ(FYFP/FCFP) = 0.0341 ± 0.0026 (n=27)) reflecting an interaction of GRK2 with the receptor. The Gβγ-binding-deficient GRK2 mutant showed a dramatically reduced FRET increase confirming the importance of Gβγ for the GRK2 recruitment to the receptor (Δ(FYFP/FCFP) = 0.0048 ± 0.0005 (n=19), p<0.05). Interestingly, the FRET development with the Gαq-binding-deficient GRK2 mutant was significantly impaired as well compared to wild-type GRK2 (Δ(FYFP/FCFP) = 0.0134 ± 0.0007 (n=15), p<0.05). This argues in favour of a contribution of Gαq to the GRK2 recruitment to the receptor. This hypothesis is supported by an agonist-dependent membrane translocation of the Gβγ-binding-deficient GRK2 that was observed in confocal microscopy. The GRK2 double mutant that does not bind the G-protein could not be recruited to the membrane. Furthermore FRET development between fluorescently labelled GRK2 and either Gαq or Gβγ reflected the interaction of GRK2 with both G-protein subunits. As expected the Gαq-binding-deficient GRK2 mutant interacted with Gβγ comparable to wild-type GRK2 (GRK2: Δ(FYFP/FCFP) = 0.41 ± 0.02 (n=21), GRK2 (D110A): Δ(FYFP/FCFP) = 0.30 ± 0.04 (n=10), not significant). Additionally, the Gβγ-binding-deficient GRK2 mutant interacted with Gαq as well (Δ(FYFP/FCFP) = 0.25 ± 0.02 (n=19)) further supporting the role of Gαq in membrane targeting of GRK2. Evaluation of kinetics by monoexponential fitting revealed a halftime of t1/2 = 0.95 s (k = 0.73 ± 0.05 s-1 (n=28)) for the Gβγ-binding by GRK2 and of t1/2 = 3.34 s (k = 0.21 ± 0.02 s-1 (n=25)) for the Gαq-binding suggesting that the GRK2 membrane targeting by Gαq is delayed compared to Gβγ.

Taken together, our results reveal an important role of Gαq in the efficient recruitment of GRK2 to the M3-ACh receptor. Gβγ-binding-deficient GRK2 mutants even demonstrate that active Gαq can cause membrane translocation of GRK2 on its own.

(1) Gurevich EV et al, Pharmacol Ther 133 (1): 40, 2012

(2) Lohse MJ et al, Pharmacol Rev 64 (2): 299, 2012

(3) Sterne-Marr R et al, J. Biol. Chem. 278 (8): 6050, 2003

(4) Carman CV et al, J. Biol. Chem. 275 (14): 10443, 2000