Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization.

The internalization of G protein-coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.

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

Most G protein-coupled receptors (GPCRs) recruit ß-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 ß-arrestin and internalization. However, the contribution of GRK2 and GRK3 to ß-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 ß-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 ß-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 ß-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.