Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells

Most G protein-coupled receptors (GPCRs) recruit {beta}-arrestins and internalize upon agonist stimulation. For the -opioid receptor (-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for -OR recruitment of {beta}-arrestin and internalization. However, the contribution of GRK2 and GRK3 to {beta}-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in {beta}-arrestin2 recruitment and -OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced -OR internalization and {beta}-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of {beta}-arrestin2 recruitment to the plasma membrane upon -OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.