New insights into non-canonical desensitization of Gq signaling by GRK2/3 expression levels

Gq-coupled receptors, upon agonist-driven activation, stimulate PLC to produce DAG and release Ca2+ stores. Desensitization of these agonist-bound receptors mediated by GRK2/3 involves sequestration of Gq to these GRKs for membrane anchoring. It has been shown that GRK2 interacts with the Gαq subunit and the βγ dimer by inserting itself between them. Increased GRK2 expression has been reported in clinical samples of heart failure and cardiac hypertrophy. Influence of GRK overexpression on Gq-signaling as well as the underlying mechanism is poorly understood. We used a Split Luciferase Complementation (SLC) system and the model Gq-coupled M1mAChR in GRK KO HEK293 cells to investigate influence of GRK overexpression on Gq-PLC interaction. GRK2 and 3 overexpression reduced Gq-PLC interaction not GRK5 and 6. Treatment with compound 101 (a GRK2/3 kinase activity inhibitor) did not have an effect in preventing GRK2 from inhibiting Gq-PLC interaction. Kinase dead mutants of GRK2/3 showed reduction in Gq-PLC interaction to the same extent as GRK2/3. This suggested that GRK2/3 mediated Gq sequestration is independent of GRK2/3 kinase activity. However, Gq-PLC interaction was rescued by using GRK mutants with reduced binding affinity to the Gαq subunit (GRK2/3(D110A) or to the βγ dimer (GRK2/3(R587Q). Furthermore, overexpressed GRK2/3 as well as kinase-dead GRK2 reduced DAG production and Ca2+ release as compared to GRK2/3 KO confirming that GRK2/3 overexpression can negatively affect Gq-initiated signaling. In conclusion, our findings suggest that PLC-β stimulation by the Gq activation is inhibited by the spatial presence of GRK2 and GRK3 and is independent of their kinase activities.