Induction of Cardiac Fibrosis by β-Blocker in G Protein-independent and G Protein-coupled Receptor Kinase 5/β-Arrestin2-dependent Signaling Pathways

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called “biased ligands” elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β₁-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β₁-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β₁-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers.     

Desensitization and Internalization of Endothelin Receptor A: IMPACT OF G PROTEIN-COUPLED RECEPTOR KINASE 2 (GRK2)-MEDIATED PHOSPHORYLATION*

Endothelin receptor A (ET_A), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ET_A mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ET_A desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gα_q coupling-deficient mutant GRK2-D110A suppressed ET_A-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ET_A-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ET_A-WT and ET_A-6PD. This demonstrates that ET_A desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gα_q and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ET_A-WT and ET_A-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ET_A-WT and the phosphorylation-deficient mutant ET_A-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: INSIGHTS FROM INTACT CELL PHOSPHORYLATION AND RECRUITMENT ASSAYS*

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α_2A-adrenergic receptors (α_2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α_2AAR with EC₅₀ values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu⁴, Val⁷, Leu⁸, Val¹¹, and Ser¹², directly interacts with receptors, whereas residues such as Asp¹⁰, Tyr¹³, Ala¹⁶, Met¹⁷, Gly⁴⁷⁵, Val⁴⁷⁷, and Ile⁴⁸⁵ are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.     

The β-blocker Nebivolol Is a GRK/β-arrestin Biased Agonist

Nebivolol, a third generation β-adrenoceptor (β-AR) antagonist (β-blocker), causes vasodilation by inducing nitric oxide (NO) production. The mechanism via which nebivolol induces NO production remains unknown, resulting in the genesis of much of the controversy regarding the pharmacological action of nebivolol. Carvedilol is another β-blocker that induces NO production. A prominent pharmacological mechanism of carvedilol is biased agonism that is independent of Gα_s and involves G protein-coupled receptor kinase (GRK)/β-arrestin signaling with downstream activation of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK). Due to the pharmacological similarities between nebivolol and carvedilol, we hypothesized that nebivolol is also a GRK/β-arrestin biased agonist. We tested this hypothesis utilizing mouse embryonic fibroblasts (MEFs) that solely express β₂-ARs, and HL-1 cardiac myocytes that express β₁- and β₂-ARs and no detectable β₃-ARs. We confirmed previous reports that nebivolol does not significantly alter cAMP levels and thus is not a classical agonist. Moreover, in both cell types, nebivolol induced rapid internalization of β-ARs indicating that nebivolol is also not a classical β-blocker. Furthermore, nebivolol treatment resulted in a time-dependent phosphorylation of ERK that was indistinguishable from carvedilol and similar in duration, but not amplitude, to isoproterenol. Nebivolol-mediated phosphorylation of ERK was sensitive to propranolol (non-selective β-AR-blocker), AG1478 (EGFR inhibitor), indicating that the signaling emanates from β-ARs and involves the EGFR. Furthermore, in MEFs, nebivolol-mediated phosphorylation of ERK was sensitive to pharmacological inhibition of GRK2 as well as siRNA knockdown of β-arrestin 1/2. Additionally, nebivolol induced redistribution of β-arrestin 2 from a diffuse staining pattern into more intense punctate spots. We conclude that nebivolol is a β₂-AR, and likely β₁-AR, GRK/β-arrestin biased agonist, which suggests that some of the unique clinically beneficial effects of nebivolol may be due to biased agonism at β₁- and/or β₂-ARs.     

Regulation of Fc?RI Signaling in Mast Cells by G Protein-coupled Receptor Kinase 2 and Its RH Domain

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on FcϵRI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2^flox/flox/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ∼50% decrease in antigen-induced Ca²⁺ mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the FcϵRI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of FcϵRIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates FcϵRI signaling in mast cells via at least two mechanisms. One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt.     

Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signaling

The serotonin (5-hydroxytryptamine; 5-HT)_2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT_2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca²⁺-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT_2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT_2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT_2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT_2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT_2C receptor agonists and their antidepressant-like activity.     

Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling

Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway.     

ERK5 Activation by Gq-Coupled Muscarinic Receptors Is Independent of Receptor Internalization and β-Arrestin Recruitment

G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.     

Type 1 Cannabinoid Receptor Ligands Display Functional Selectivity in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

Modulation of type 1 cannabinoid receptor (CB₁) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB₁ are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC)) on arrestin2-, Gα_i/o-, Gβγ-, Gα_s-, and Gα_q-mediated intracellular signaling in the mouse STHdh^Q7/Q7 cell culture model of striatal medium spiny projection neurons that endogenously express CB₁. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα_i/o and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB₁ levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα_q-dependent pathways. CP55,940 and CBD both signaled through Gα_s. CP55,940, but not CBD, activated downstream Gα_s pathways via CB₁ targets. THC and CP55,940 promoted CB₁ internalization and decreased CB₁ protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB₁ leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.     

Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors

Activation of cannabinoid CB1 receptors (CB₁R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT_2A receptors (5-HT_2AR) revealed a remarkable 5-HT_2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT_2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB₁R and 5-HT_2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT_2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB₁R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB₁R and 5-HT_2AR mediating cognitive impairment. CB₁R-5-HT_2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.     

Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction: FORMATION OF THE CANNABINOID CB2 RECEPTOR-Gi PROTEIN COMPLEX*

In this study, we applied a comprehensive G protein-coupled receptor-Gα_i protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB₂)- Gα_i interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gα_i C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gα_i C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gα_i α₄β₆ loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB₂·G_i complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB₂ R*·Gα_i1β₁γ₂ complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gα_sβ₁γ₂ complex crystal structure, the Gα_i1β₁γ₂ protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gα_i1 α5 helix tilt changed due to the outward movement of TMH5 in CB₂ R*. 2) A 25° clockwise rotation of Gα_i1β₁γ₂ underneath CB₂ R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gα_i1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to G_i proteins.     

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β₂-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling.     

Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands

The cannabinoid receptor 1 (CB₁) and CB₂ cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a β-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing β-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CβII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CβII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy.The CB₁² and CB₂ cannabinoid receptors comprise a two-member subfamily of G-protein-coupled receptors (GPCRs) that are notable as the targets of the tetrahydrocannabinol (THC) derivatives found in marijuana. More recently CB₁ receptors along with other GPCRs have been promoted as therapeutic pharmacological targets in the billion dollar weight loss market for controversial drugs such as rimonabant (SR141716A) and Fen-phen. Thus, an important utility of cannabinoid family receptors to society appears to arise from their role in regulating a broad spectrum of addiction-based behaviors, and the addition of new members to the cannabinoid receptor family may have social and economic implications that reach far beyond the initial scientific discovery. As a consequence, the re-classification of an orphan GPCR as a cannabinoid family member should be done with caution requiring strict criteria of receptor activation by THC derivatives or endogenous cannabinoid compounds and a widespread agreement of the results by the scientific community.Marijuana, one of the most widely abused substances (1), mediates many of its psychotropic effects by targeting CB₁ receptors in the central nervous system, but studies with CB₁ and CB₂ knock-out mice indicate that the complex pharmacological properties on pain, mood, and memory exhibited by exogenous cannabinoids and the endogenous arachidonic acid-based endo-cannabinoids, including anandamide and 2-arachidonoylglycerol (2-AG), are not fully explained by their activation of CB₁ and CB₂ (2–4). The CB₁ and CB₂ receptors are 44% identical and signal through G_i/o-mediated pathways. Activation of either receptor is inhibitory for cAMP production via adenylyl cyclase and stimulatory for mitogen-activated protein kinase (MAPK) (extracellular-regulated protein kinase 1/2 (ERK1/2)) activation (5). However, the failure of these two receptors to account for the full complement of physiological effects observed with cannabinoid ligands has led to the hypothesis that additional cannabinoid-like receptors exist.The orphan GPCR, GPR55, which exhibits only 10–15% homology to the two human cannabinoid receptors (6), is one of a number of plausible cannabinoid family member candidates (7). GPR55 was first identified and mapped to human chromosome 2q37 a decade ago (8). In the human central nervous system, it is predominantly localized to the caudate, putamen, and striatum (8), coupling to Gα₁₃ (9, 10), Gα₁₂, or Gα_q (11).GPR55 has been tested against a number of cannabinoid ligands with mixed results. Observations using a GTPγS functional assay indicate that GPR55 is activated by nanomolar concentrations of the endocannabinoids 2-AG, virodhamine, noladin ether, and palmitoylethanolamine (10) and the atypical cannabinoids Abn-CBD and O-1602 (12) as well as by the drugs CP55,950, HU210, and Δ⁹-THC (11). Exposure of GPR55 to the cannabinoids THC and JWH015 in dorsal root ganglion neurons and in receptor-transfected HEK293 cells correlates with increases of intracellular Ca²⁺ (11). In contrast, GPR55 is insensitive to the CB₁ inverse agonist AM281 and the potent cannabinoid agonist WIN55212-2 but is antagonized by the marijuana constituent CBD (9, 10). However, Oka et al. (13) reported that GPR55 is not a typical cannabinoid receptor, as numerous endogenous and synthetic cannabinoids, including many mentioned above, had no effect on GPR55 activity. They present compelling data suggesting that the endogenous lipid LPI and its 2-arachidonyl analogs are agonists at GPR55 as a result of their abilities to phosphorylate extracellular-regulated kinase and induce calcium signaling (13, 14). Further studies indicate that LPI and the rimonabant-like CB₁ inverse agonist AM251 induce oscillatory Ca²⁺ release through Gα₁₃ and RhoA (9). These reports were all performed in HEK 293 cells, yet each documented a distinct and conflicting chemical space of agonists that recognized GPR55. To resolve these inconsistencies in classification, an alternative approach for identifying GPR55 ligands that is insensitive to the endogenous complement of cellular receptors could circumvent many of the challenges that have arisen in the measurements of G-protein signaling.β-Arrestins are intracellular proteins that bind and desensitize activated GPCRs and in the process form stable receptor/arrestin signaling complexes (15, 16). β-Arrestin redistribution to the activated membrane-bound receptor represents one of the early intracellular events provoked by agonist binding and, consequently, is less prone to a false positive or negative readout as compared with studying a downstream signaling event as a readout of receptor activation. β-arrestin-green fluorescent chimeras can make this process attractive to monitor by forming remarkably sensitive and specific probes of GPCR activation that are independent of downstream G-protein-mediated signaling (17–19). We have determined GPR55 responsiveness to a representative panel of cannabinoid ligands and LPI in the presence (and absence) of a β-arrestin2-green fluorescent protein (βarr2-GFP) biosensor. Our data demonstrate that LPI, the CB₁ inverse agonist/antagonists SR141716A, and AM251 are GPR55 agonists, and the CB₁ agonist CP55940 is a GPR55 antagonist/partial agonist. These data together with our inability to observe activation of GPR55 by Δ⁹-THC and endocannabinoids indicate that GPR55 should be classified as an atypical cannabinoid receptor at best.     

Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB₁ cannabinoid receptors (CB₁R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB₁R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB₁R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227–235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB₁R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB₁R mRNA and protein, the most efficacious being the RARγ agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB₁R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARγ to the CB₁R gene 5′ upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB₁R expression was attenuated by small interfering RNA knockdown of RARγ in hepatocytes. We conclude that RARγ regulates CB₁R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.     

Substitution of 5-HT1A Receptor Signaling by a Light-activated G Protein-coupled Receptor

Understanding serotonergic (5-HT) signaling is critical for understanding human physiology, behavior, and neuropsychiatric disease. 5-HT mediates its actions via ionotropic and metabotropic 5-HT receptors. The 5-HT_1A receptor is a metabotropic G protein-coupled receptor linked to the G_i/o signaling pathway and has been specifically implicated in the pathogenesis of depression and anxiety. To understand and precisely control 5-HT_1A signaling, we created a light-activated G protein-coupled receptor that targets into 5-HT_1A receptor domains and substitutes for endogenous 5-HT_1A receptors. To induce 5-HT_1A-like targeting, vertebrate rhodopsin was tagged with the C-terminal domain (CT) of 5-HT_1A (Rh-CT_5-HT1A). Rh-CT_5-HT1A activates G protein-coupled inward rectifying K⁺ channels in response to light and causes membrane hyperpolarization in hippocampal neurons, similar to the agonist-induced responses of the 5-HT_1A receptor. The intracellular distribution of Rh-CT_5-HT1A resembles that of the 5-HT_1A receptor; Rh-CT_5-HT1A localizes to somatodendritic sites and is efficiently trafficked to distal dendritic processes. Additionally, neuronal expression of Rh-CT_5-HT1A, but not Rh, decreases 5-HT_1A agonist sensitivity, suggesting that Rh-CT_5-HT1A and 5-HT_1A receptors compete to interact with the same trafficking machinery. Finally, Rh-CT_5-HT1A is able to rescue 5-HT_1A signaling of 5-HT_1A KO mice in cultured neurons and in slices of the dorsal raphe showing that Rh-CT_5-HT1A is able to functionally compensate for native 5-HT_1A. Thus, as an optogenetic tool, Rh-CT_5-HT1A has the potential to directly correlate in vivo 5-HT_1A signaling with 5-HT neuron activity and behavior in both normal animals and animal models of neuropsychiatric disease.     

Functional Selectivity of 6′-Guanidinonaltrindole (6′-GNTI) at κ-Opioid Receptors in Striatal Neurons

There is considerable evidence to suggest that drug actions at the κ-opioid receptor (KOR) may represent a means to control pain perception and modulate reward thresholds. As a G protein-coupled receptor (GPCR), the activation of KOR promotes Gα_i/o protein coupling and the recruitment of β-arrestins. It has become increasingly evident that GPCRs can transduce signals that originate independently via G protein pathways and β-arrestin pathways; the ligand-dependent bifurcation of such signaling is referred to as “functional selectivity” or “signaling bias.” Recently, a KOR agonist, 6′-guanidinonaltrindole (6′-GNTI), was shown to display bias toward the activation of G protein-mediated signaling over β-arrestin2 recruitment. Therefore, we investigated whether such ligand bias was preserved in striatal neurons. Although the reference KOR agonist U69,593 induces the phosphorylation of ERK1/2 and Akt, 6′-GNTI only activates the Akt pathway in striatal neurons. Using pharmacological tools and β-arrestin2 knock-out mice, we show that KOR-mediated ERK1/2 phosphorylation in striatal neurons requires β-arrestin2, whereas Akt activation depends upon G protein signaling. These findings reveal a point of KOR signal bifurcation that can be observed in an endogenous neuronal setting and may prove to be an important indicator when developing biased agonists at the KOR.     

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB₁ receptor activity. Here we investigated the possibility of a negative regulation by CB₁ receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB₁-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL^−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB₁ activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.     

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB₁ cannabinoid receptors (CB₁Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB₁R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB₁Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB₁Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.