GRK Screening Assays

G protein-coupled receptors (GPCRs) play a central role in how cells respond to external signals. Their activity is controlled by a small group of enzymes called GPCR kinases (GRKs), specifically GRK2, GRK3, GRK5, and GRK6, which are present in nearly all cell types. In several diseases, including cancer and heart failure, individual GRKs are produced at abnormally high levels. This has increased interest in developing drugs that can selectively inhibit these enzymes. Although many GRK inhibitors have been studied using computer-based models or biochemical test systems, only a limited number of approaches allow their effectiveness to be tested directly in living cells. To overcome this limitation, we developed a robust cell-based assay using engineered HEK293 cells. These cells express the β2-adrenergic receptor together with a single GRK isoform, while all four endogenous GRKs have been removed. This setup enables precise analysis of how individual GRKs contribute to receptor phosphorylation. Using a well-established 7-transmembrane (7TM) phosphorylation assay, we measured receptor phosphorylation triggered by the β-agonist isoproterenol. This platform allowed us to systematically evaluate the performance of commercially available GRK inhibitors in a physiologically relevant cellular context.