Abstract
Most G protein-coupled receptors (GPCRs) recruit β-arrestins and are internalized upon agonist stimulation. For the μ-opioid receptor (μ-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 μ-OR recruitment of β-arrestin and internalization. However, the contribution of GRK2 and GRK3 to β-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. By CRISPR/Cas9 we established HEK293 cells with knock-out of GRK2, GRK3 or both to dissect their individual contributions in β-arrestin2 recruitment and μ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced μ-OR internalization substantially. Furthermore, we found GRK2 to be more important for μ-OR internalization than GRK3. In contrast, the effect of GRK2/3 knock-out on β-arrestin2 recruitment was minor. Rescue expression experiments restored GRK2/3 functions. The GRK2/3 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.