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Dissecting mGlu5 receptor internalization pathways using genetic and pharmacological tools

Periodic Reporting for period 1 - INTERGLU (Dissecting mGlu5 receptor internalization pathways using genetic and pharmacological tools)

Reporting period: 2018-11-01 to 2020-10-31

G protein-coupled receptors (GPCRs) are membrane proteins that are activated by a broad range of ligands and among the most abundant gene families in the human genome. GPCRs are involved in many important physiological processes and are the target of 30-40% of all marketed drugs. The most important function of GPCRs is to activate intracellular signaling cascades that regulate cellular function. Activation of intracellular partners is regulated by receptor internalization to avoid overstimulation of the cell. The metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR that plays a key role in synaptic plasticity, generally thought to be the basis of learning and memory formation. Thus, it is not surprising that mGlu5 is a promising drug target for several central nervous system disorders, including Fragile X syndrome, Parkinson’s disease, addictive disorders, anxiety and schizophrenia.

The receptor internalization mechanism has been well-characterized for a few GPCRs and a generalized model has been developed. However, the receptor internalization pathway has not been studied for the majority of GPCRs and it remains unclear whether they follow the canonical pathway. In fact, there is an increasing number of studies suggesting that non-canonical receptor internalization is more prevalent than previously anticipated.

The overall objectives of the INTERGLU project (grant agreement number 797497) were: (1) to develop novel genetic and pharmacological tools to study GPCR internalization and (2) to apply them to dissect the internalization mechanisms of GPCRs and in particular the mGlu5 receptor.
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).
µOR is the main mediator of both the beneficial effects and the side effects of opioid drugs. Desensitization and internalization are believed to play important roles in developing opioid tolerance and GRKs have previously been implicated in this process. Using our GRK knockout cells, we could clearly delineate the dependence of the µOR internalization and arrestin recruitment on the individual GRKs. This could explain cell type differences due to differences in GRK expression. The knockout cells constitute an important addition to the toolbox available to study GPCR desensitization, arrestin recruitment and internalization because the cells provide complete and specific knockout of individual GRKs, exemplified by our study with the µOR.

We expect our work with delineation of the arrestin and G protein dependence of GPCR internalization and the related review to lead to a re-examination of the canonical, arrestin-centric model of GPCR internalization and further studies of the alternative internalization pathways. In particular the arrestin-independent pathways, about which little is known.

The pharmacological characterization of the mGlu5 PAMs and NAMs is the first to include the internalization pathway and it could provide insights into future design of drugs modulating mGlu5 activity.
Delineating G protein-coupled receptor internalization pathways using knockout cell lines