Knockout cell lines were developed using the CRISPR/Cas9 technology targeting the GPCR kinase (GRK) family. GRKs play a critical role in the internalization of many GPCRs by phosphorylating intracellular domains of the receptor which allows for binding of arrestin or other proteins that internalize the receptor. We developed cell lines with knockout of GRK2, GRK3 and GRK2/3. The developed GRK knockout cell lines were used to determine the GRK dependence of the µ-opioid receptor (µOR) internalization and arrestin recruitment. We showed that µOR is mainly depending on GRK2 for internalization and arrestin recruitment and identified a GRK-independent, sustained component of the arrestin recruitment to the plasma membrane. Interestingly, this component was not associated with µOR. The characterization of the cells and the functional study with µOR was published in Scientific Reports (Møller, Pedersen et al., Scientific Reports, 2020. 10: 17395).
We used cells with knockout of arrestins and G proteins and G protein inhibitors to study the internalization of mGlu5 and several other GPCRs. A manuscript describing these findings is currently in preparation. Related to this study, we wrote a review with focus on the arrestin-independent GPRC internalization pathways that is currently undergoing peer review.
The internalization pathways of mGlu5 (and other GPCRs) could depend on how it is activated, also called biased agonism. Ligands that bind to another site than the endogenous ligand, called allosteric modulators, likely have an increased potential of activating the receptor in a different way than ligands binding to the endogenous ligand binding site. In addition, they provide subtype selectivity. This is important for the metabotropic glutamate receptors, where 8 subtypes exist that in some cases have opposing functions. We studied how mGlu5 internalization and signaling was affected by nine allosteric modulators that have an inhibitory effect on ligands binding to the endogenous ligand binding site, called negative allosteric modulators (NAMs). All of the NAMs inhibited internalization and we identified inhibition biased towards or away from the internalization pathway for four of the NAMs. The results of the NAM study were published in Molecular Pharmacology (Arsova et al. Molecular Pharmacology, 2020. 98(1): 49-60). We also studied the effect of three positive allosteric modulators (PAMs) on mGlu5 internalization and signaling. This study is currently undergoing peer review.
As part of the studies of GPCR internalization in knockout cell lines and with pharmacological inhibitors of internalization, the researcher co-authored several publications where he contributed with knowledge of GPCR internalization (Leach et al. Pharmacological Reviews, 2020. 72(3): 558-604) and/or expertise in measuring GPCR internalization (Sundqvist et al., Journal of Immunology, 2019. 203(12): 3349-3360; Sundqvist et al., Biochimica et Biophysica Acta – Molecular Cell Research, 2020. 1867(12): 118849; Pedersen et al., Neuropharmacology, 2020. 166: 107718).
