TY - JOUR

T1 - Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells

AU - Møller, Thor C

AU - Pedersen, Mie F

AU - van Senten, Jeffrey R

AU - Seiersen, Sofie D

AU - Mathiesen, Jesper M

AU - Bouvier, Michel

AU - Bräuner-Osborne, Hans

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

U2 - 10.1038/s41598-020-73674-0

DO - 10.1038/s41598-020-73674-0

M3 - Journal article

C2 - 33060647

VL - 10

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 17395

ER -