Table of Contents:
G-protein-coupled receptor kinase type 2 (GRK2) belongs to the family of serine/threonine-kinases and plays an important role in the desensitisation of G-protein-coupled receptors (GPCR). Its agonist-dependent recruitment to activated GPCRs through interaction with free Gβγ-subunits and negatively charged phospholipids of the plasma membrane has been studied in detail (Pitcher et al., 1992; Touhara et al., 1995). In addition, Gαq was identified to be an interaction partner of GRK2 (Carman et al., 1999b). Thus, GRK2 can be considered to be a G protein effector that sequesters active Gq-protein subunits and thereby desensitises GPCRs in a phosphorylation-independent manner. It remained unclear, whether – and if so which – additional effects on the signal transduction of Gq-protein-coupled receptors might result from the interaction between Gαq and GRK2. Furthermore, there was no detailed description of the kinetics of the agonist-mediated recruitment of GRK2 to Gq-protein-coupled receptors. Neither were any kinetics data available for the dissociation of receptor and GRK2 after agonist withdrawal.
The aim of this study was to uncover the dynamics of the interaction of GRK2 with Gq protein-coupled receptors and other proteins involved in the GRK2-recruitment. The M3 acetylcholine (ACh) receptor was chosen as a model receptor. In this respect, the influence of Gαq binding to GRK2 was investigated. Therefore, experiments based on Fluorescence Resonance Energy Transfer (FRET) were established. This technique allowed the visualisation and investigation of the interaction between GRK2 and M3-ACh receptors, Gαq, and Gβγ-subunits, respectively, in single living HEK293T-cells with high spatio-temporal resolution. Stimulation of M3-ACh receptors with acetylcholine resulted in distinct FRET-changes that reflected the interaction of the labelled partner. GRK2-Mutants with reduced affinity to Gαq or Gβγ were used to analyse the influence of Gq-protein binding to GRK2. So far, the role of Gβγ in membrane translocation of GRK2 had only been studied using end-point experiments. This study could confirm the published results using the newly established FRET-assays and other fluorescence-imaging techniques, while also providing for the first time kinetic details. It was further observed that the agonist-dependent membrane targeting of GRK2 is not only mediated by Gβγ, but also by Gαq, thereby uncovering a new aspect of GRK2 membrane translocation. Furthermore, Gαq increases extent and stability of the interaction of GRK2 and the M3-ACh receptor. This fact was revealed by comparison of the absolute FRET-amplitudes of different GRK2-mutants. Investigation of the kinase activity of GRK2 showed that binding of Gβγ is indeed a necessary prerequisite for receptor phosphorylation, but efficient receptor phosphorylation is only achieved through simultaneous binding of Gβγ and Gαq to GRK2. These results further confirmed the importance of Gαq for the recruitment of GRK2 to activated M3-ACh receptors, although Gβγ has a larger effect in this respect. However, GRK2 shows higher affinity towards Gαq than Gβγ, which is indicated by the fact that the interaction between GRK2 and Gαq is more sensitive than the GRK2-Gβγ-interaction. Accordingly, Gαq is presumably more important for the GRK2-recruitment at lower agonist concentrations. No effect of activated Gq-protein subunits was observed on the GRK2-recruitment towards Gi-protein-coupled receptors during these studies. Therefore, the observed influence of Gαq is probably limited to Gq-protein-coupled receptors. The FRET-based experiments allowed for the first time the detailed description of the kinetics of GRK2-recruitment to the M3-ACh receptor, beginning with the interaction between activated receptor and inactive Gq-proteins and ending at the complex formation of GRK2 with Gαq, Gβγ and the receptor. It became evident that the binding between GRK2 and Gβγ occurred about 3-times as fast as the respective interaction of GRK2 with Gαq. The membrane translocation of GRK2, mediated through binding to Gq-proteins, is the rate-limiting step for the interaction of GRK2 with the receptor. The interaction between GRK2 and M3-ACh receptors was prolonged by Gαq. This hints at an inhibition of receptor signalling straight after GRK2 binding and not just after recruitment of arrestin, as assumed previously.
Taken together, this work shows for the first time the importance of Gαq in the translocation of GRK2 towards M3-ACh receptors, thereby exerting critical influence on signal transduction and desensitisation of Gq-protein-coupled receptors. Furthermore, this study also provides the first detailed description of the kinetics of all steps involved in this physiologically important pathway.