PT  - JOURNAL ARTICLE
AU  - Valerie Wolters
AU  - Cornelius Krasel
AU  - Joerg Brockmann
AU  - Mortiz Bunemann
TI  - Influence of Gαq on the Dynamics of M3-ACh-receptor-GRK2 Interaction
AID  - 10.1124/mol.114.094722
DP  - 2014 Jan 01
TA  - Molecular Pharmacology
PG  - mol.114.094722
4099  - http://molpharm.aspetjournals.org/content/early/2014/10/10/mol.114.094722.short
4100  - http://molpharm.aspetjournals.org/content/early/2014/10/10/mol.114.094722.full
AB  - G-protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase with an important function in the desensitisation of G-protein-coupled receptors. Based on its ability to bind G-protein βγ subunits as well as activated Gαq subunits it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well-known to be mediated by Gβγ together with negatively charged membrane phospholipids. In the current study we address the role of Gαq on the interaction of GRK2 with activated Gq-protein-coupled receptors. Therefore, we established new FRET-based assays to study the interaction of GRK2 with the M3-ACh receptor as well as Gq-protein subunits with high spatio-temporal resolution in single living HEK293T cells. M3-ACh receptor stimulation with 10 μM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding-affinity towards Gαq (GRK2(D110A)) and Gβγ (GRK2(R587Q)) were utilised in order to determine the specific role of Gq-protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gαq enhances the extent and stability of the GRK2-M3-ACh receptor interaction and that not only Gβγ but also Gαq can target GRK2 to the membrane. This reveals an important role of Gαq in efficient recruitment of GRK2 to M3-ACh receptors. Furthermore, interactions between Gαq and GRK2 were associated with a prolongation of the interaction between GRK2 and the M3-ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Gαq influences signalling and desensitisation.