Most G protein-coupled receptors (GPCRs) recruit β-arrestins and internalize 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. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout 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 and β-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 β-arrestin2 recruitment to the plasma membrane upon μ-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.

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14Most G protein-coupled receptors (GPCRs) recruit b-arrestins and are internalized upon agonist 15 stimulation. For the µ-opioid receptor (µ-OR), this process has been linked to development of opioid 16 tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for 17 µ-OR recruitment of b-arrestin and internalization. However, the contribution of GRK2 and GRK3 to 18 b-arrestin recruitment and receptor internalization, remain to be determined in their complete 19 absence. By CRISPR/Cas9 we established HEK293 cells with knock-out of GRK2, GRK3 or both to 20 dissect their individual contributions in b-arrestin2 recruitment and µ-OR internalization upon 21 stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced 22 µ-OR internalization substantially. Furthermore, we found GRK2 to be more important for µ-OR 23 internalization than GRK3. In contrast, the effect of GRK2/3 knock-out on b-arrestin2 recruitment 24 was minor. Rescue expression experiments restored GRK2/3 functions. The GRK2/3 small molecule 25 inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, 26 cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 27concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR 28 function. 29 30 Introduction: 31The family of G protein-coupled receptors (GPCRs) constitute important drug targets, through 32 which ~30% of all clinically approved medicines mediate their action 1 . Regulation of GPCR signaling 33 following receptor activation is a complex process that typically involves recruitment of kinases that 34 phosphorylate the receptor, which increases the affinity for b-arrestins and mediate receptor 35 internalization 2 . The µ-opioid receptor (µ-OR) belongs to the family of rhodopsin-like (family A) 36 3 GPCRs and mediates the analgesic effects of opioid drugs and related side-effects and addictive 37 properties 3,4 . The regulation of µ-OR desensitization and trafficking has been suggested to be linked 38 to the development of tolerance after chronic use of opioid drugs 5 and it is therefore important to 39 understand the underlying molecular mechanisms. 40Several studies indicate that receptor phosphorylation by GPCR kinases (GRKs) is important for 41 the desensitization of µ-OR signaling and initiation of internalization [6][7][8][9] . Out of the seven isotypes in 42 the GRK family, four (GRK2, GRK3, GRK5 and GRK6) have been speculated to regulate µ-OR in vivo 43 due to their overlapping expression patterns 10 . Knock-out (KO) models have confirmed the 44 importance of the individual GRKs. For instance, it has been demonstrated that fentanyl and 45 morphine-induced tolerance are decreased in GRK3 KO mice 11 , morphine reward and dependence 46 are lost in mice depleted of GRK5, but not GRK3, and morphine-induced locomotor activity is 47 increased in mice lacking GRK6 compared to wild type littermates 12,13 . Altoget...