Biased Signaling of the Mu Opioid Receptor Revealed in Native Neurons

Ehrlich, Aliza T.; Semache, Meriem; Gross, Florence; Fonte, Dillon F. Da; Runtz, Léonie; Colley, Christine; Mezni, Amina; Gouill, Christian Le; Lukasheva, Viktoriya; Hogue, Mireille; Darcq, Emmanuel; Bouvier, Michel; Kieffer, Brigitte L.

doi:10.1016/j.isci.2019.03.011

Cited by 70 publications

(81 citation statements)

References 35 publications

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“…When considering βarrestin recruitment to the PM upon receptor stimulation, our analysis shows that 22% of receptors did not recruit βarrestin beyond the established threshold, even in the presence of overexpressed GRK2, which is known to favor recruitment (Ehrlich et al, 2019) (Figure 4E). Among the receptors able to recruit arrestins, only a very small number of receptors selectively recruited either βarrestin1 (1.3%) or βarrestin2 (6.4%), most of them recruiting both βarrestin subtypes in the presence of GRK2 (92.3%) ( Figure 4E).…”

Section: Diversity Of Coupling Selectivity Between G Protein Familiesmentioning

confidence: 94%

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

Avet

Mancini

Breton

et al. 2020

Preprint

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The ability of individual G protein-coupled receptors (GPCR) to engage multiple signaling pathways opens opportunities for the development of better drugs. This requires new knowledge and tools to determine the G protein subtypes and arrestins engaged by a given receptor. Here, we used a new BRET-based effector membrane translocation assay (EMTA) that monitors activation of each Gα protein through the recruitment of selective G protein effectors and βarrestins to the plasma membrane. Profiling of 100 therapeutically relevant GPCR revealed a great diversity of coupling profiles with some receptors displaying exquisite selectivity, whereas others promiscuitely engage all four G protein families. Comparison with existing datasets points to commonalities but also to critical differences between studies.Combining a biosensor subset allowed detecting activity of nearly all GPCR thus providing a new tool for safety screens and systems pharmacology. Overall, this work describes unique resources for studying GPCR function and drug discovery. KEYWORDSG protein-coupled receptor (GPCR), enhanced bystander bioluminescence resonance energy transfer (ebBRET), Biosensor, Effector membrane translocation assay (EMTA), Highthroughput assay, G protein activation, Functional selectivity, Systems pharmacology.

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Section: Diversity Of Coupling Selectivity Between G Protein Familiesmentioning

confidence: 94%

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

Avet

Mancini

Breton

et al. 2020

Preprint

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“…The involvement of µ-OR phosphorylation in these processes was confirmed by later studies 10,[21][22][23][24] . In addition to GRK2, in vitro studies have shown that GRK3/5/6 have direct roles in µ-OR phosphorylation and/or internalization 16,23,[25][26][27] . Moreover, studies have also utilized phospho-site specific antibodies to demonstrate that the µ-OR is differentially phosphorylated by distinct GRK isotypes depending on the agonist used.…”

Section: Introductionmentioning

confidence: 99%

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

Pedersen

Møller

Seiersen

et al. 2020

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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...

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“…It is well-established that MOR regulation is strongly agonist dependent and it can be argued that in the present study, we used morphine, which has low affinity for β-arresin-2, to demonstrate that opioid tolerance in the small intestine is β-arrestin-2-independent. However, we have previously shown that tolerance in the isolated ileum develops to repeated exposure of high efficacy opioid agonists like fentanyl or etorphine, known to engage β-arrestins with high affinity (McPherson et al, 2010;Maguma et al, 2012;Ehrlich et al, 2019). Consequently, the β-arrestin-2-independent mechanism of tolerance in the ileum is not contingent on the opioid agonist.…”

Section: Discussionmentioning

confidence: 99%

Rapid tolerance to morphine in the myenteric neurons of the small intestine is independent of β-arrestin-2 and mediated by PKC

Muchhala

Jacob

Alam

et al. 2020

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The differential rates of opioid tolerances raise the question of discrete underlying mechanisms. While opioid tolerance is strongly linked to the development of dependence, there is a dissociation between the two phenomena in the gut as tolerance does not develop to chronic opioid-induced constipation, but diarrhea still manifests upon withdrawal. Here, we find that tolerance develops to inhibition of small intestinal motility after one day of morphine exposure, and is more rapid compared to spinally-mediated antinociception and constipation in chronic morphine-treated mice. This tolerance can be reversed by protein kinase C (PKC) inhibition but not by β-arrestin-2 deletion. Similarly, morphine tolerance develops to inhibition of neuronal excitability in ileum myenteric neurons independently of β-arrestin-2 and is attenuated by inhibiting PKC. These findings reveal a potential mechanism for differences in the rates of tolerances to opioids and implicate myenteric neurons of the ileum as the primary cause for opioid-induced withdrawal effects

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Biased Signaling of the Mu Opioid Receptor Revealed in Native Neurons

Cited by 70 publications

References 35 publications

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

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

Rapid tolerance to morphine in the myenteric neurons of the small intestine is independent of β-arrestin-2 and mediated by PKC

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