Linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse neural tube development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing the primary cilium, the cell's antenna, which acts as a signaling hub. Fuz, an effector of planar cell polarity signaling, regulates Shh signaling by facilitating cilia formation, and the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of the genes encoding these proteins is similar; however, their functional relationship has not been previously explored. This study identified the genetic and biochemical linkage between Fuz and Gpr161 in mouse neural tube development. Fuz was found to be genetically epistatic to Gpr161 with respect to regulation of Shh signaling in mouse neural tube development. The Fuz protein biochemically interacts with Gpr161, and Fuz regulates Gpr161-mediated ciliary localization, a process that might utilize ß-arrestin 2. Our study characterizes a previously unappreciated Gpr161-Fuz axis that regulates Shh signaling during mouse neural tube development.

α-linolenic acid mitigates microglia-mediated neuroinflammation of schizophrenia in mice by suppressing the NF-κB/NLRP3 pathway via binding GPR120-ß-arrestin 2.

BACKGROUND: Schizophrenia (SCZ) is a heterogeneous psychiatric disorder that is poorly treated by current therapies. Emerging evidence indicates that SCZ is closely correlated with a persistent neuroinflammation. α-linolenic acid (ALA) is highly concentrated in the brain and represents a modulator of the immune system by decreasing the inflammatory response in chronic metabolic diseases. This study was first designed to investigate the potential role of dietary ALA on cognitive function and neuroinflammation in mice with SCZ. METHODS: In vivo, after 2 weeks of modeling, mice were treated with dietary ALA treatment for 6 weeks. In vitro, inflammation model was created using lipopolysaccharide as an inducer in BV2 microglial cells. RESULTS: Our results demonstrated that ALA alleviated cognitive impairment and enhanced synaptic plasticity in mice with SCZ. Moreover, ALA mitigated systematic and cerebral inflammation through elevating IL-10 and inhibiting IL-1ß, IL-6, IL-18 and TNF-α. Furthermore, ALA notably inhibited microglia and pro-inflammatory monocytes, as well as microglial activation andpolarization. Mechanistically, ALA up-regulated the expressions of G protein coupled receptor (GPR) 120 and associated ß-inhibitor protein 2 (ß-arrestin2), accompanied by observable weakened levels of transforming growth factor-ß activated kinase 1 (TAK1), NF-κB p65, cysteine proteinase-1 (caspase-1), pro-caspase-1, associated speck-like protein (ASC) and NLRP3. In vitro, ALA directly restrained the inflammation of microglia by decreasing the levels of pro-inflammatory factors and regulating microglial polarization via GPR120-NF-κB/NLRP3inflammasome signaling pathway, whereas AH7614 definitely eliminated this anti-inflammatory effect of ALA. CONCLUSION: Dietary ALA ameliorates microglia-mediated neuroinflammation by suppressing the NF-κB/NLRP3 pathway via binding GPR120-ß-arrestin2.

Molecular mechanism of ß-arrestin-2 pre-activation by phosphatidylinositol 4,5-bisphosphate.

Phosphorylated residues of G protein-coupled receptors bind to the N-domain of arrestin, resulting in the release of its C-terminus. This induces further allosteric conformational changes, such as polar core disruption, alteration of interdomain loops, and domain rotation, which transform arrestins into the receptor-activated state. It is widely accepted that arrestin activation occurs by conformational changes propagated from the N- to the C-domain. However, recent studies have revealed that binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the C-domain transforms arrestins into a pre-active state. Here, we aimed to elucidate the mechanisms underlying PIP2-induced arrestin pre-activation. We compare the conformational changes of ß-arrestin-2 upon binding of PIP2 or phosphorylated C-tail peptide of vasopressin receptor type 2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Introducing point mutations on the potential routes of the allosteric conformational changes and analyzing these mutant constructs with HDX-MS reveals that PIP2-binding at the C-domain affects the back loop, which destabilizes the gate loop and ßXX to transform ß-arrestin-2 into the pre-active state.

Myeloid beta-arrestin 2 depletion attenuates metabolic dysfunction-associated steatohepatitis via the metabolic reprogramming of macrophages.

Macrophage-mediated inflammation has been implicated in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH); however, the immunometabolic program underlying the regulation of macrophage activation remains unclear. Beta-arrestin 2, a multifunctional adaptor protein, is highly expressed in bone marrow tissues and macrophages and is involved in metabolism disorders. Here, we observed that ß-arrestin 2 expression was significantly increased in the liver macrophages and circulating monocytes of patients with MASH compared with healthy controls and positively correlated with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD). Global or myeloid Arrb2 deficiency prevented the development of MASH in mice. Further study showed that ß-arrestin 2 acted as an adaptor protein and promoted ubiquitination of immune responsive gene 1 (IRG1) to prevent increased itaconate production in macrophages, which resulted in enhanced succinate dehydrogenase activity, thereby promoting the release of mitochondrial reactive oxygen species and M1 polarization. Myeloid ß-arrestin 2 depletion may be a potential approach for MASH.

Key phosphorylation sites for robust ß-arrestin2 binding at the MOR revisited.

Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, phosphorylation is important for ß-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser is the primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr get subsequently and rapidly phosphorylated. Here we used GRK KO cells, phosphosite specific antibodies and site-directed mutagenesis to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and ß-arrestin2 recruitment. We show that both GRK2/3 and GRK5/6 subfamilies promote phosphorylation of 370Thr and 375Ser. Importantly, only GRK2/3 induce phosphorylation of 376Thr and 379Thr, and we identify these residues as key sites to promote robust ß-arrestin recruitment to the MOR. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of GRK subfamilies plays an important role in determining the tissue responses of opioid agonists.

A molecular mechanism for angiotensin II receptor blocker-mediated slit membrane protection: Angiotensin II increases nephrin endocytosis via AT1-receptor-dependent ERK 1/2 activation.

Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Ang II enhances albuminuria by ß-Arrestin2-mediated nephrin endocytosis. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-ß-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-ß-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and ß-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and ß-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin ß-Arrestin2 binding. The mutation of ß-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. ß-Arrestin2K11R,K12R does not influence nephrin cell surface expression. The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria.

Insight into the regulatory mechanism of ß-arrestin2 and its emerging role in diseases.

ß-arrestin2, a member of the arrestin family, mediates the desensitization and internalization of most G protein-coupled receptors (GPCRs) and functions as a scaffold protein in signalling pathways. Previous studies have demonstrated that ß-arrestin2 expression is dysregulated in malignant tumours, fibrotic diseases, cardiovascular diseases and metabolic diseases, suggesting its pathological roles. Transcription and post-transcriptional modifications can affect the expression of ß-arrestin2. Furthermore, post-translational modifications, such as phosphorylation, ubiquitination, SUMOylation and S-nitrosylation affect the cellular localization of ß-arrestin2 and its interaction with downstream signalling molecules, which further regulate the activity of ß-arrestin2. This review summarizes the structure and function of ß-arrestin2 and reveals the mechanisms involved in the regulation of ß-arrestin2 at multiple levels. Additionally, recent studies on the role of ß-arrestin2 in some major diseases and its therapeutic prospects have been discussed to provide a reference for the development of drugs targeting ß-arrestin2.

Activation of µ receptors by SR-17018 through a distinctive mechanism.

Agonists at µ opioid receptors relieve acute pain, however, their long-term use is limited by side effects, which may involve ß-arrestin2. Agonists biased against ß-arrestin2 recruitment may be advantageous. However, the classification of bias may be compromised by assays utilising overexpressed µ receptors which overestimate efficacy for G-protein activation. There is a need for re-evaluation with restricted receptor availability to determine accurate agonist efficacies. We depleted µ receptor availability in PathHunter CHO cells using the irreversible antagonist, ß-funaltrexamine (ß-FNA), and compared efficacies and apparent potencies of twelve agonists, including several previously reported as biased, in ß-arrestin2 recruitment and cAMP assays. With full receptor availability all agonists had partial efficacy for stimulating ß-arrestin2 recruitment relative to DAMGO, while only TRV130 and buprenorphine were partial agonists as inhibitors of cAMP accumulation. Limiting receptor availability by prior exposure to ß-FNA (100 nM) revealed morphine, oxycodone, PZM21, herkinorin, U47700, tianeptine and U47931e are also partial agonists in the cAMP assay. The efficacies of all agonists, except SR-17018, correlated between ß-arrestin2 recruitment and cAMP assays, with depleted receptor availability in the latter. Furthermore, naloxone and cyprodime exhibited non-competitive antagonism of SR-17018 in the ß-arrestin2 recruitment assay. Limited antagonism by naloxone was also non-competitive in the cAMP assay, while cyprodime was competitive. Furthermore, SR-17018 only negligibly diminished ß-arrestin2 recruitment stimulated by DAMGO (1 µM), whereas fentanyl, morphine and TRV130 all exhibited the anticipated competitive inhibition. The data suggest that SR-17018 achieves bias against ß-arrestin2 recruitment through interactions with µ receptors outside the orthosteric agonist site. This article is part of the Special Issue on "Ligand Bias".

Inverse Regulation of C-C Chemokine Receptor 3 Oligomerization by Downstream Proteins Indicates Biased Signal Transduction Pathways.

Oligomerization is one of the important mechanisms for G protein-coupled receptors (GPCRs) to modulate their activity in signal transduction. However, details of how and why the oligomerization of GPCRs regulates their functions under physiological conditions remain largely unknown. Here, using single-molecule photobleaching technology, we show that chemokine ligand 5 (CCL5) and chemokine ligand 8 (CCL8) are similar to the previously reported chemokine ligand 11 (CCL11) and chemokine ligand 24 (CCL24), which can regulate the oligomerization of chemokine receptor 3 (CCR3). Our results further demonstrate that downstream proteins, ß-arrestin 2 and Gi protein complex, on the CCR3 signal transduction pathway, can inversely regulate the oligomeric states of CCR3 induced by its binding ligands. This unexpected discovery suggests complex relationships between the oligomeric behaviors of CCR3 and the components of ligands-CCR3-downstream proteins, reflecting the potentially functional impact of the oligomerization on the multiple activation pathways of GPCR, such as biased activation.

Notoginsenoside R1 attenuates ischemic heart failure by modulating MDM2/ß arrestin2-mediated ß2-adrenergic receptor ubiquitination.

ß2 adrenergic receptor (ß2AR) is a G-protein-coupled receptor involved in cardiac protection. In chronic heart failure (CHF), persistent sympathetic nervous system activation occurs, resulting in prolonged ß2AR activation and subsequent receptor desensitization and downregulation. Notoginsenoside R1 (NGR1) has the functions of enhancing myocardial energy metabolism and mitigating myocardial fibrosis. The mechanisms of NGR1 against ischemic heart failure are unclear. A left anterior descending (LAD) artery ligation procedure was performed on C57BL/6 J mice for four weeks. From the 4th week onwards, they were treated with various doses (3, 10, 30 mg/kg/day) of NGR1. Subsequently, the impacts of NGR1 on ischemic heart failure were evaluated by assessing cardiac function, morphological changes in cardiac tissue, and the expression of atrial natriuretic peptide (ANP) and beta-myosin heavy chain (ß-MHC). H9c2 cells were protected by NGR1 when exposed to OGD/R conditions. H9c2 cells were likewise protected from OGD/R damage by NGR1. Furthermore, NGR1 increased ß2AR levels and decreased ß2AR ubiquitination. Mechanistic studies revealed that NGR1 enhanced MDM2 protein stability and increased the expression of MDM2 and ß-arrestin2 while inhibiting their interaction. Additionally, under conditions produced by OGD/R, the protective benefits of NGR1 on H9c2 cells were attenuated upon administration of the MDM2 inhibitor SP141. According to these findings, NGR1 impedes the interplay between ß-arrestin2 and MDM2, thereby preventing the ubiquitination and degradation of ß2AR to improve CHF.

ß-arrestin2 is indispensable for the antidepressant effects of fluoxetine via inhibiting astrocytic pyroptosis in chronic mild stress mouse model for depression.

ß-arrestin2 is a versatile protein for signaling transduction in brain physiology and pathology. Herein, we investigated the involvement of ß-arrestin2 in pharmacological effects of fluoxetine for depression. A chronic mild stress (CMS) model was established using wild-type (WT) and ß-arrestin2-/- mice. Behavioral results demonstrated that CMS mice showed increased immobility time in the tail suspension test and forced swimming test, elevated concentrations of pro-inflammatory factors in peripheral blood, increased expression of pyroptosis-related proteins, and increased co-labeling of glial fibrillary acidic protein and Caspase1 p10 in the hippocampus compared to the CON group. Treatment with fluoxetine (FLX) ameliorated these conditions. However, compared with the ß-arrestin2-/- CMS group, these results of the ß-arrestin2-/- CMS + FLX group showed no significant changes. These results suggested that the above effects of FLX could be eliminated by knocking out ß-arrestin2. Mass spectrometry implying that FLX promoted the binding of ß-arrestin2 to the NLRP2 inflammasome of depressed mice. Subsequently, the results of the cellular experiments suggested that the 5HT2B receptor antagonist may attenuate L-kynurenine + ATP-induced cell pyroptosis by attenuating NLRP2 binding to ß-arrestin2. We further found that the lack of ß-arrestin2 eliminated the anti-pyroptosis effect of fluoxetine. In conclusion, ß-arrestin2 is an essential protein for fluoxetine to alleviate pyroptosis in the hippocampal astrocytes of CMS mice. Mechanistically, we found that the 5-HT2BR-ß-arrestin2-NLRP2 axis is vital for maintaining the antidepressant effects of fluoxetine.

Characterization of genetic variants of GIPR reveals a contribution of ß-arrestin to metabolic phenotypes.

Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes1. Yet both activation and inhibition of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in combination with glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) activation have resulted in similar clinical outcomes, as demonstrated by the GIPR-GLP-1R co-agonist tirzepatide2 and AMG-133 (ref. 3) combining GIPR antagonism with GLP-1R agonism. This underlines the importance of a better understanding of the GIP system. Here we show the necessity of ß-arrestin recruitment for GIPR function, by combining in vitro pharmacological characterization of 47 GIPR variants with burden testing of clinical phenotypes and in vivo studies. Burden testing of variants with distinct ligand-binding capacity, Gs activation (cyclic adenosine monophosphate production) and ß-arrestin 2 recruitment and internalization shows that unlike variants solely impaired in Gs signalling, variants impaired in both Gs and ß-arrestin 2 recruitment contribute to lower adiposity-related traits. Endosomal Gs-mediated signalling of the variants shows a ß-arrestin dependency and genetic ablation of ß-arrestin 2 impairs cyclic adenosine monophosphate production and decreases GIP efficacy on glucose control in male mice. This study highlights a crucial impact of ß-arrestins in regulating GIPR signalling and overall preservation of biological activity that may facilitate new developments in therapeutic targeting of the GIPR system.

Loss of ß-arrestin2 aggravated condylar cartilage degeneration at the early stage of temporomandibular joint osteoarthritis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative joint disorder characterized by extracellular matrix degeneration and inflammatory response of condylar cartilage. ß-arrestin2 is an important regulator of inflammation response, while its role in TMJOA remains unknown. The objective of this study was to investigate the role of ß-arrestin2 in the development of TMJOA at the early stage and the underlying mechanism. METHODS: A unilateral anterior crossbite (UAC) model was established on eight-week-old wild-type (WT) and ß-arrestin2 deficiency mice to simulate the progression of TMJOA. Hematoxylin-eosin (HE) staining and microcomputed tomography (micro-CT) analysis were used for histological and radiographic assessment. Immunohistochemistry was performed to detect the expression of inflammatory and degradative cytokines, as well as autophagy related factors. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay was carried out to assess chondrocyte apoptosis. RESULTS: The loss of ß-arrestin2 aggravated cartilage degeneration and subchondral bone destruction in the model of TMJOA at the early stage. Furthermore, in UAC groups, the expressions of degradative (Col-X) and inflammatory (TNF-α and IL-1ß) factors in condylar cartilage were increased in ß-arrestin2 null mice compared with WT mice. Moreover, the loss of ß-arrestin2 promoted apoptosis and autophagic process of chondrocytes at the early stage of TMJOA. CONCLUSION: In conclusion, we demonstrated for the first time that ß-arrestin2 plays a protective role in the development of TMJOA at the early stage, probably by inhibiting apoptosis and autophagic process of chondrocytes. Therefore, ß-arrestin2 might be a potential therapeutic target for TMJOA, providing a new insight for the treatment of TMJOA at the early stage.

Positive allosteric modulation of the cannabinoid CB1 receptor potentiates endocannabinoid signalling and changes ERK1/2 phosphorylation kinetics.

BACKGROUND AND PURPOSE: Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation. EXPERIMENTAL APPROACH: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and ß-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA. KEY RESULTS: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed. CONCLUSION AND IMPLICATIONS: Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including ß-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

TRAF6-mediated ubiquitination of AKT1 in the nucleus occurs in a ß-arrestin2-dependent manner upon insulin stimulation.

AKT, also known as protein kinase B (PKB), serves as a crucial regulator of numerous biological functions, including cell growth, metabolism, and tumorigenesis. Increasing evidence suggests that the kinase activity of AKT is regulated via ubiquitination by various E3 ligase enzymes in response to different stimuli. However, the molecular mechanisms underlying insulin-induced AKT ubiquitination are not yet fully understood. Here, we show that activation of the insulin receptor (IR) leads to enhanced ubiquitination of AKT1 at K8 and K14 residues, facilitated by the cytosolic E3 ubiquitin ligase enzyme, TRAF6. Further investigation using AKT1 mutants with modified nucleocytoplasmic shuttling properties reveals that TRAF6-mediated AKT1 ubiquitination occurs within the nucleus in a ß-Arr2-dependent manner. The nuclear entry of TRAF6 depends on importin ß1, while ß-Arr2 regulates this process by facilitating the interaction between TRAF6 and importin ß1. Additionally, the ubiquitination of AKT1 is essential for its translocation to the activated IR on the plasma membrane, where it plays a functional role in recruiting Glut4 and facilitating glucose uptake. This study uncovers the cellular components and processes involved in insulin-induced ubiquitination and activation of AKT1, providing insights and detailed strategies for manipulating AKT1.

ArreSTick motif controls ß-arrestin-binding stability and extends phosphorylation-dependent ß-arrestin interactions to non-receptor proteins.

The binding and function of ß-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established ß-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with ß-arrestins can be identified, a pattern that we name "arreSTick." Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and ß-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by ß-arrestin2. Our findings unveil a broader role for ß-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.

Investigation of the intrinsic cannabinoid activity of hemp-derived and semisynthetic cannabinoids with ß-arrestin2 recruitment assays-and how this matters for the harm potential of seized drugs.

Cultivation of industrial low-Δ9-tetrahydrocannabinol (Δ9-THC) hemp has created an oversupply of cannabidiol (CBD)-rich products. The fact that phytocannabinoids, including CBD, can be used as precursors to synthetically produce a range of THC variants-potentially located in a legal loophole-has led to a diversification of cannabis recreational drug markets. 'Hemp-compliant', 'hemp-derived' and 'semisynthetic' cannabinoid products are emerging and being advertised as (legal) alternatives for Δ9-THC. This study included a large panel (n = 30) of THC isomers, homologs, and analogs that might be derived via semisynthetic procedures. As a proxy for the abuse potential of these compounds, we assessed their potential to activate the CB1 cannabinoid receptor with a ß-arrestin2 recruitment bioassay (picomolar-micromolar concentrations). Multiple THC homologs (tetrahydrocannabihexol, THCH; tetrahydrocannabiphorol, THCP; tetrahydrocannabinol-C8, THC-C8) and THC analogs (hexahydrocannabinol, HHC; hexahydrocannabiphorol, HHCP) were identified that showed higher potential for CB1 activation than Δ9-THC, based on either higher efficacy (Emax) or higher potency (EC50). Structure-activity relationships were assessed for Δ9-THC and Δ8-THC homologs encompassing elongated alkyl chains. Additionally, stereoisomer-specific differences in CB1 activity were established for various THC isomers (Δ7-THC, Δ10-THC) and analogs (HHC, HHCP). Evaluation of the relative abundance of 9(S)-HHC and 9(R)-HHC epimers in seized drug material revealed varying epimeric compositions between batches. Increased abundance of the less active 9(S)-HHC epimer empirically resulted in decreased potency, but sustained efficacy for the resulting diastereomeric mixture. In conclusion, monitoring of semisynthetic cannabinoids is encouraged as the dosing and the relative composition of stereoisomers can impact the harm potential of these drugs, relative to Δ9-THC products.

Mapping the interaction site for ß-arrestin-2 in the prokineticin 2 receptor.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the ß-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with ß-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to ß-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

Genetic Deletion of ß-Arrestin 2 From the Subfornical Organ and Other Periventricular Nuclei in the Brain Alters Fluid Homeostasis and Blood Pressure.

BACKGROUND: ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the AT1R [angiotensin type 1 receptor])-mediated AT1R in the subfornical organ. Recently, we demonstrated that ARRB2 (ß-arrestin 2) global knockout mice exhibit a higher preference for salt and exacerbated pressor response to deoxycorticosterone acetate salt. However, whether ARRB2 within selective neuroanatomical nuclei alters physiological responses to ANG is unknown. Therefore, we hypothesized that ARRB2, specifically in the subfornical organ, counterbalances maladaptive dipsogenic and pressor responses to the canonical AT1R signaling. METHODS: Male and female Arrb2FLOX mice received intracerebroventricular injection of either adeno-associated virus (AAV)-Cre-GFP (green fluorescent protein) to induce brain-specific deletion of ARRB2 (Arrb2ICV-Cre). Arrb2FLOX mice receiving ICV-AAV-GFP were used as control (Arrb2ICV-Control). Infection with ICV-AAV-Cre primarily targeted the subfornical organ with few off targets. Fluid intake was evaluated using the 2-bottle choice paradigm with 1 bottle containing water and 1 containing 0.15 mol/L NaCl. RESULTS: Arrb2ICV-Cre mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, Arrb2ICV-Cre mice exhibited a significant increase in saline intake compared with controls, resulting in a saline preference. Furthermore, when mice were subjected to water-deprived or sodium-depleted conditions, which would naturally increase endogenous ANG levels, Arrb2ICV-Cre mice exhibited elevated saline intake. CONCLUSIONS: Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the subfornical organ, counterbalances canonical AT1R responses to both exogenous and endogenous ANG. Stimulation of the AT1R/ARRB axis in the brain may represent a novel strategy to treat hypertension.

GPCR kinase subtype requirements for arrestin-2 and -3 translocation to the cannabinoid CB1 receptor and the consequences on G protein signalling.

Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB1 receptor (CB1) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB1, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB1 must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.