Abstract Body: Introduction: Beneficial cardiac effects of G-protein-coupled receptor kinase 2 (GRK2) inhibition in heart failure are attributed to resensitization of the impaired beta-adrenoceptor-stimulated cAMP response.
Research Question: We asked whether we could establish a stable cellular model which detects GRK2 and GRK2 inhibition effects on cAMP signaling stimulated by beta(1)-adrenoceptors (ADRB1).
Aims: The study aimed to elucidate GRK2 effects on ADRB(1)-stimulated cAMP signaling in real-time using EPAC-based cAMP biosensors.
Methods: The following HEK cell lines with expression of EPAC-based cAMP sensors (EPAC) were established: (a) Control EPAC cells, (b) EPAC cells with GRK2 overexpression, and (c) EPAC cells with expression of GRK2-inhibitory, GRK2K220R (K220R). Signaling by ADRB1 was determined in (d) ADRB1-EPAC cells expressing a beta1-adrenoceptor-EPAC fusion protein, (e) ADRB1-EPAC cells with GRK2 overexpression, and (f) ADRB1-EPAC cells with K220R expression.
Results: Cell stimulation with isoproterenol triggered the typical transient cAMP increase. EC50 values of the isoproterenol-stimulated cAMP signal were not significantly different between EPAC cells with endogenous GRK2, and with GRK2 or K220R overexpression. In contrast, the maximum cAMP peak height after isoproterenol stimulation was significantly reduced in GRK2-overexpressing EPAC cells by 22.3 ± 7.6 % compared to EPAC cells with endogenous GRK2 and by 36.1 ± 6.3 % compared to K220R-expressing cells (mean ± s.d., n=3). The reduced cAMP peak in GRK2-expressing EPAC cells was attributed to the kinase activity of GRK2 because the GRK2-specific kinase inhibitor, CMPD101, increased the cAMP peak only in GRK2-expressing EPAC cells. HEK cells have endogenous expression of ADRB1 and ADRB2. To detect specifically ADRB1-stimulated cAMP signaling, we generated an ADRB1-EPAC fusion protein. GRK2 expression significantly reduced the ADRB1-EPAC-mediated cAMP peak height by 23.7 ± 7.8 % compared to ADRB1-EPAC control cells whereas K220R had no significant effect on the ADRB1-EPAC stimulated signal (mean ± s.d.; n = 6; p = 0.0059; one-way ANOVA, Tukey’s test)
Conclusion: We established a cellular system which detects kinase activity-mediated GRK2 effects specifically on ADRB1-stimulated cAMP signaling.