Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s).

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.

Chronic loss of noradrenergic tone produces ß-arrestin2-mediated cocaine hypersensitivity and alters cellular D2 responses in the nucleus accumbens.

Cocaine blocks plasma membrane monoamine transporters and increases extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT). The addictive properties of cocaine are mediated primarily by DA, while NE and 5-HT play modulatory roles. Chronic inhibition of dopamine ß-hydroxylase (DBH), which converts DA to NE, increases the aversive effects of cocaine and reduces cocaine use in humans, and produces behavioral hypersensitivity to cocaine and D2 agonism in rodents, but the underlying mechanism is unknown. We found a decrease in ß-arrestin2 (ßArr2) in the nucleus accumbens (NAc) following chronic genetic or pharmacological DBH inhibition, and overexpression of ßArr2 in the NAc normalized cocaine-induced locomotion in DBH knockout (Dbh -/-) mice. The D2/3 agonist quinpirole decreased excitability in NAc medium spiny neurons (MSNs) from control, but not Dbh -/- animals, where instead there was a trend for an excitatory effect. The Gαi inhibitor NF023 abolished the quinpirole-induced decrease in excitability in control MSNs, but had no effect in Dbh -/- MSNs, whereas the Gαs inhibitor NF449 restored the ability of quinpirole to decrease excitability in Dbh -/- MSNs, but had no effect in control MSNs. These results suggest that chronic loss of noradrenergic tone alters behavioral responses to cocaine via decreases in ßArr2 and cellular responses to D2/D3 activation, potentially via changes in D2-like receptor G-protein coupling in NAc MSNs.

ß-Arrestin1 enhances hepatocellular carcinogenesis through inflammation-mediated Akt signalling.

G-protein-coupled receptors (GPCR) constitute the largest known superfamily for signal transduction and transmission, and they control a variety of physiological and pathological processes. GPCR adaptor ß-arrestins (ARRBs) play a role in cancerous proliferation. However, the effect of ARRBs in inflammation-mediated hepatocellular carcinogenesis is unknown. Here we show that ARRB1, but not ARRB2, is upregulated in inflammation-associated hepatocellular carcinoma (HCC) and paracancerous tissues in humans. A genotoxic carcinogen, diethylnitrosamine (DEN), significantly induces hepatic inflammation, TNF-α production and ARRB1 expression. Although ARRB1 deficiency does not affect hepatic inflammation and TNF-α production, it markedly represses hepatocellular carcinogenesis by suppressing malignant proliferation in DEN-treated mice. Furthermore, TNF-α directly induces hepatic ARRB1 expression and enhances ARRB1 interaction with Akt by binding to boost Akt phosphorylation, resulting in malignant proliferation of liver cells. Our data suggest that ARRB1 enhances hepatocellular carcinogenesis by inflammation-mediated Akt signalling and that ARRB1 may be a potential therapeutic target for HCC.

Muscarinic acetylcholine receptor M3 modulates odorant receptor activity via inhibition of ß-arrestin-2 recruitment.

The olfactory system in rodents serves a critical function in social, reproductive and survival behaviours. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odour-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odours. Here, we show that an M3-R antagonist attenuates odour-induced responses in OSNs from wild-type, but not M3-R-null, mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of ß-arrestin-2 to ORs, resulting in a potentiation of odour-induced responses in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

Discovery of 4-(phenyl)thio-1H-pyrazole derivatives as agonists of GPR109A, a high affinity niacin receptor.

Even though nicotinic acid (niacin) appears to have beneficial effects on human lipid profiles, niacin-induced cutaneous vasodilatation called flushing limits its remedy to patient. GPR109A is activated by niacin and mediates the anti-lipolytic effects. Based on the hypothesis that ß-arrestin signaling mediates niacin-induced flushing, but not its anti-lipolytic effect, we tried to find GPR109A agonists which selectively elicit Gi-protein-biased signaling devoid of ß-arrestin internalization using a ß-lactamase assay. We identified a 4-(phenyl)thio-1H-pyrazole as a novel scaffold for GPR109A agonist in a high throughput screen, which has no carboxylic acid moiety known to be important for binding. While 1-nicotinoyl derivatives (5a-g, 6a-e) induced ß-arrestin recruitment, 1-(pyrazin-2-oyl) derivatives were found to play as G-protein-biased agonists without GPR109A receptor internalization. The activity of compound 5a (EC50 = 45 nM) was similar to niacin (EC50 = 52 nM) and MK-6892 (EC50 = 74 nM) on calcium mobilization assay, but its activity at 10 µM on ß-arrestin recruitment were around two and five times weaker than niacin and MK-6892, respectively. The development of G-protein biased GPR109A ligands over ß-arrestin pathway is attainable and might be important in differentiation of pharmacological efficacy.

Indoloditerpenes from a marine-derived fungal strain of Dichotomomyces cejpii with antagonistic activity at GPR18 and cannabinoid receptors.

A marine-derived strain of Dichotomomyces cejpii produces the new compounds emindole SB beta-mannoside (1) and 27-O-methylasporyzin C (2), as well as the known indoloditerpenes JBIR-03 (3) and emindole SB (4). Indole derivative 1 was found to be a CB2 antagonist, while 2 was identified as the first selective GPR18 antagonist with an indole structure. Compound 4 was found to be a nonselective CB1/CB2 antagonist. The new natural indole derivatives may serve as lead structures for the development of GPR18- and CB receptor-blocking drugs.

FTY720 (s)-phosphonate preserves sphingosine 1-phosphate receptor 1 expression and exhibits superior barrier protection to FTY720 in acute lung injury.

OBJECTIVE: Effective therapies are needed to reverse the increased vascular permeability that characterizes acute inflammatory diseases such as acute lung injury. FTY720 is a pharmaceutical analog of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate. Because both FTY720 and sphingosine 1-phosphate have properties that may limit their usefulness in patients with acute lung injury, alternative compounds are needed for therapeutic use. The objective of this study is to characterize the effects of FTY720 (S)-phosphonate, a novel analog of FTY720-phosphate, on variables of pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo. SETTING: University-affiliated research institute. SUBJECTS: Cultured human pulmonary endothelial cells; C57BL/6 mice. INTERVENTIONS: Endothelial cells were stimulated with sphingosine 1-phosphate receptor 1 agonists to determine effects on sphingosine 1-phosphate receptor 1 expression. Acute lung injury was induced in C57BL/6 mice with bleomycin to assess effects of sphingosine 1-phosphate receptor 1 agonists. MEASUREMENTS AND MAIN RESULTS: FTY720 (S)-phosphonate potently increases human pulmonary endothelial cell barrier function in vitro as measured by transendothelial electrical resistance. Reduction of sphingosine 1-phosphate receptor 1 with small interference RNA significantly attenuates this transendothelial electrical resistance elevation. FTY720 (S)-phosphonate maintains endothelial sphingosine 1-phosphate receptor 1 protein expression in contrast to greater than 50% reduction after incubation with sphingosine 1-phosphate, FTY720, or other sphingosine 1-phosphate receptor 1 agonists. FTY720 (S)-phosphonate does not induce ß-arrestin recruitment, sphingosine 1-phosphate receptor 1 ubiquitination, and proteosomal degradation that occur after other agonists. Intraperitoneal administration of FTY720 (S)-phosphonate every other day for 1 week in normal or bleomycin-injured mice maintains significantly higher lung sphingosine 1-phosphate receptor 1 expression compared with FTY720. FTY720 fails to protect against bleomycin-induced acute lung injury in mice, while FTY720 (S)-phosphonate significantly decreases lung leak and inflammation. CONCLUSION: FTY720 (S)-phosphonate is a promising barrier-promoting agent that effectively maintains sphingosine 1-phosphate receptor 1 levels and improves outcomes in the bleomycin model of acute lung injury.

Phthalate is associated with insulin resistance in adipose tissue of male rat: role of antioxidant vitamins.

Diethyl hexyl phthalate (DEHP) is a plasticizer, commonly used in a variety of products, including lubricants, perfumes, hairsprays and cosmetics, construction materials, wood finishers, adhesives, floorings and paints. DEHP is an endocrine disruptor and it has a continuum of influence on various organ systems in human beings and experimental animals. However, specific effects of DEHP on insulin signaling in adipose tissue are not known. Adult male albino rats of Wistar strain were divided into four groups. Control, DEHP treated (dissolved in olive oil at a dose of 10, and 100 mg/kg body weight, respectively, once daily through gastric intubations for 30 days) and DEHP + vitamin E (50 mg/kg body weight) and C (100 mg/kg body weight) dissolved in olive oil and distilled water, respectively, once daily through gastric intubations for 30 days. After the completion of treatment, adipose tissue was dissected out to assess various parameters. DEHP treatment escalated H(2)O(2) and hydroxyl radical levels as well as lipid peroxidation in the adipose tissue. DEHP impaired the expression of insulin signaling molecules and their phosphorelay pathways leading to diminish plasma membrane GLUT4 level and thus decreased glucose uptake and oxidation. Blood glucose level was elevated as a result of these changes. Supplementation of vitamins (C & E) prevented the DEHP-induced changes. It is concluded that DEHP-induced ROS and lipid peroxidation disrupts the insulin signal transduction in adipose tissue and favors glucose intolerance. Antioxidant vitamins have a protective role against the adverse effect of DEHP.

Prostaglandin E2 promotes lung cancer cell migration via EP4-betaArrestin1-c-Src signalsome.

Many human cancers express elevated levels of cyclooxygenase-2 (COX-2), an enzyme responsible for the biosynthesis of prostaglandins. Available clinical data establish the protective effect of COX-2 inhibition on human cancer progression. However, despite these encouraging outcomes, the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer drugs. Hence, a better understanding of the molecular signals downstream of COX-2 is needed for the elucidation of drug targets that may improve cancer therapy. Here, we show that the COX-2 product prostaglandin E(2) (PGE(2)) acts on cognate receptor EP4 to promote the migration of A549 lung cancer cells. Treatment with PGE(2) enhances tyrosine kinase c-Src activation, and blockade of c-Src activity represses the PGE(2)-mediated lung cancer cell migration. PGE(2) affects target cells by activating four receptors named EP1 to EP4. Use of EP subtype-selective ligand agonists suggested that EP4 mediates prostaglandin-induced A549 lung cancer cell migration, and this conclusion was confirmed using a short hairpin RNA approach to specifically knock down EP4 expression. Proximal EP4 effectors include heterotrimeric Gs and betaArrestin proteins. Knockdown of betaArrestin1 expression with shRNA significantly impaired the PGE(2)-induced c-Src activation and cell migration. Together, these results support the idea that increased expression of the COX-2 product PGE(2) in the lung tumor microenvironment may initiate a mitogenic signaling cascade composed of EP4, betaArrestin1, and c-Src which mediates cancer cell migration. Selective targeting of EP4 with a ligand antagonist may provide an efficient approach to better manage patients with advanced lung cancer.

Beta-arrestin signaling complex as a target for antidepressants and as a depression marker.

Beta-arrestins uncouple G protein-coupled receptors (GPCRs) from G proteins and promote their internalization, leading to desensitization and downregulation and serving as negative regulators of GPCR signaling. beta-Arrestins also function as scaffold proteins, interacting with several cytoplasmic proteins and linking GPCRs to intracellular signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade. Recent work has also revealed that beta-arrestins translocate from the cytoplasm to the nucleus and associate with transcription factors such as histone acetyltransferase p300 and cyclic adenosine monophosphate (cAMP)-responsive element-binding protein. These substances also interact with regulators of transcription factors. We review findings on the effects of antidepressants on beta-arrestins and the plethora of antidepressant effects on signal transduction elements in which beta-arrestins serve as signaling scaffold proteins, focusing on the three major groups of MAPKs: extracellular signal-regulated kinases, c-Jun N-terminal kinases and p38 MAPKs, and on transcription factors and cofactors of which beta-arrestins mediate transcription regulation.

C5a- and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I-C5L2 mutation.

UNLABELLED: C5L2, a G-protein-coupled receptor (GPCR), has been identified as an ASP (C3adesArg) and C5a receptor. Controversy exists regarding both ligand binding and functionality. ASP activation of C5L2 is proposed to regulate fat storage. C5L2 is also proposed as a decoy receptor for C5a, an inflammatory mediator, based on absence of Ca(2+) or chemotaxis changes. AIMS: (i) to evaluate C5L2 receptor activation and recycling using recombinant ASP (rASP) and rC5a and (ii) assess receptor trafficking of S323I-C5L2 mutation previously identified in a family and demonstrated to have altered functionality. RESULTS: stably transfected C5L2-HEK cells were sorted using fluorescent-ASP (Fluos-ASP) binding. Following 2-h serum-free pretreatment, C5L2 was typically localized to the cell-surface. beta-Arrestin-2-GFP transiently transfected C5L2-HEK cells demonstrated rASP and rC5a-dependent beta-arrestin-2-GFP translocation, which showed time-dependent intracellular colocalization with C5L2. Without ligand or C5L2 transfection, no translocation was identified at any time point. Ligand-dependent (rASP and rC5a) C5L2 endocytosis was time-dependent with a 1-h nadir, and was clathrin- and cholesterol-dependent. Transiently transfected Rab-GFP proteins (Rabs 5, 7 and 11) demonstrated time-dependent colocalization of Rab5, Rab7, and Rab11 with C5L2. In contrast to C5L2, a large proportion of stably transfected S323I-C5L2 did not localize to the cell-surface. While S323I-C5L2 was competent for Fluos-ASP and (125)I-ASP binding, although at a reduced level, there was no ligand-mediated receptor phosphorylation. Further, there was no ligand-mediated activation of beta-arrestin-2-GFP translocation, and no downstream functional activation of glucose transport or triglyceride synthesis. CONCLUSION: C5L2 is a functional metabolic receptor, and serine 323 is important for ASP induced functionality.

Antidepressants, beta-arrestins and GRKs: from regulation of signal desensitization to intracellular multifunctional adaptor functions.

G protein-coupled receptors (GPCR) have generated considerable interest in the pharmaceutical industry as drug targets. Theories concerning antidepressant targets of action suggested pre-synaptic monoamine reuptake mechanisms regulating GPCR activities including delayed receptor desensitization and down-regulation. GRKs and beta-arrestins translocate to the cell membrane and bind to agonist-occupied receptors. This uncouples these receptors from G proteins and promotes their internalization, leading to desensitization and down-regulation. Thus, GRKs and beta-arrestins serve as negative regulators of GPCR signaling. Recently, GPCR have been demonstrated to elicit signals through interaction with beta-arrestin as scaffolding proteins, independent of heterotrimeric G-protein coupling. beta-arrestins function as scaffold proteins that interact with several cytoplasmic proteins and link GPCR to intracellular signaling pathways such as MAPK cascades. Recent work has also revealed that beta-arrestins translocate from the cytoplasm to the nucleus and associate with transcription cofactors such as p300 and CREB. They also interact with regulators of transcription factors. We review findings concerning effects of antidepressants on GRKs and beta-arrestins and the plethora of antidepressants effects on signal transduction elements in which GRKs and beta-arrestins serve as signaling scaffold proteins, and on transcription factors and cofactors in which beta-arrestins mediate regulation of transcription. The emergence of G-protein-independent signaling pathways, through beta-arrestins, changes the way in which GPCR signaling is evaluated, from a cell biological to a pharmaceutical perspective and raises the possibility for the development of pathway specific therapeutics e.g., antidepressant medications targeting GRKs and beta-arrestin regulatory and signaling proteins.

Arrestins as multi-functional signaling adaptors.

Arrestins are versatile regulators of cellular signaling expressed in every cell in the body. Arrestins bind active phosphorylated forms of their cognate G-protein-coupled receptors, shutting down G-protein activation and linking receptors to alternative signaling pathways. Arrestins directly interact with more than 20 surprisingly diverse proteins, such as several Src family kinases, ubiquitin ligases, protein phosphatases, microtubules, etc., and serve as scaffolds facilitating signaling in two MAP kinase cascades, leading to the activation of ERK1/2 and JNK3. A number of arrestin-binding partners are key players in signaling pathways that regulate cell proliferation, survival, and apoptotic death, which make arrestin interactions with these proteins inviting targets for therapeutic intervention. For example, enhancement of pro-survival or pro-apoptotic arrestin-dependent signaling is a promising strategy in treating disorders such as neurodegenerative diseases or cancer, respectively. Recent studies show that in the cell arrestin exists in at least three distinct conformations, free, receptor-bound, and microtubule-bound, with very different signaling capabilities. Precise identification of arrestin elements mediating its interactions with each partner and elucidation of conformational dependence of these interactions will pave the way to the development of molecular tools for targeted enhancement or attenuation of arrestin interactions with individual partners. This structural information is necessary to devise conventional drug-based approaches and to engineer specialized "designer" arrestins that can compensate for defects in receptor regulation associated with congenital disorders and/or redirect arrestin-mediated signaling to desired pathways. Arrestins are at the crossroads of crucial pathways that determine cell fate and behavior. Therefore, targeted manipulation of arrestin-dependent signaling has an enormous therapeutic potential.

G protein coupled receptors as drug targets: the role of beta-arrestins.

G protein coupled receptors (GPCRs) are extremely important drug targets and the beta-arrestin intracellular scaffolding and adaptor proteins regulate major aspects of their pharmacology. beta-arrestin binding to activated, GPCR kinase (GRK)-phosphorylated receptors has the capacity to terminate G protein coupling, internalize the receptors into clathrin-coated vesicles and establish a secondary signaling complex independent of G protein signaling. These events appear to be differentially regulated by GRK phosphorylation, ubiquitination and potentially beta-arrestin oligomerization, which are likely to be highly receptor and cell-type dependent. The role of beta-arrestins in switching from G-protein dependent to independent signaling places them in a pivotal position to dictate the downstream effects of ligand binding. Consequently, we must appreciate the functioning of these molecules as we strive to discover and optimize new GPCR drug therapies for endocrine, metabolic and immune disorders.

A beta-arrestin 2 signaling complex mediates lithium action on behavior.

Besides their role in desensitization, beta-arrestin 1 and 2 promote the formation of signaling complexes allowing G protein-coupled receptors (GPCR) to signal independently from G proteins. Here we show that lithium, a pharmacological agent used for the management of psychiatric disorders such as bipolar disorder, schizophrenia, and depression, regulates Akt/glycogen synthase kinase 3 (GSK3) signaling and related behaviors in mice by disrupting a signaling complex composed of Akt, beta-arrestin 2, and protein phosphatase 2A. When administered to beta-arrestin 2 knockout mice, lithium fails to affect Akt/GSK3 signaling and induce behavioral changes associated with GSK3 inhibition as it does in normal animals. These results point toward a pharmacological approach to modulating GPCR function that affects the formation of beta-arrestin-mediated signaling complexes.

Characterization of isoprenaline- and salmeterol-stimulated interactions between beta2-adrenoceptors and beta-arrestin 2 using beta-galactosidase complementation in C2C12 cells.

beta-Arrestin is an adaptor protein that has been shown to couple G protein-coupled receptors (GPCRs) to clathrin-coated pits and target them for subsequent internalization. More recently, beta-arrestin 2 has also been shown to be involved in the activation of mitogen-activated protein kinase cascades by G protein-coupled receptors independently of G protein activation. Direct interactions between proteins can be monitored using enzyme complementation between two inactive deletion mutants of beta-galactosidase (beta-gal; Deltaalpha and Deltaomega). In the present study, we have used fusion proteins of the human beta(2)-adrenoceptor (C-terminal beta-gal Deltaalpha) and beta-arrestin 2 (beta-gal Deltaomega) to study directly the pharmacology of this interaction in C2C12 cells expressing the beta(2)-adrenoceptor-beta-gal Deltaalpha fusion protein at low physiological levels (38.2 +/- 2.7 fmol . mg protein(-1)). Isoprenaline, noradrenaline, and adrenaline (-log EC(50) = 5.9, 5.5, and 5.7, respectively) stimulated an association between the beta(2)-adrenoceptor and beta-arrestin 2 at much higher concentrations than required for activation of cAMP accumulation (-log EC(50) = 7.6, 6.3, and 7.7, respectively). This was sensitive to inhibition by the beta(2)-adrenoceptor antagonists propranolol, timolol, and ICI 118551. Both salbutamol and terbutaline behaved as partial agonists of beta-gal complementation. Furthermore, the long-acting beta(2)-agonist salmeterol (-log K(D) for inhibition of [(3)H]CGP12177 binding = 8.7) behaved as an antagonist of isoprenaline-stimulated beta(2)-adrenoceptor-arrestin 2 interactions (-log K(D) = 8.0), whereas acting as a full agonist of cAMP accumulation in the same cells (-log EC(50) = 9.2). These data suggest that salmeterol can discriminate between receptor-G(S) protein and receptor-arrestin 2 complexes (in terms of efficacy and affinity) in a way that is favorable for its long duration of action.

Beta-arrestin-1 levels: reduced in leukocytes of patients with depression and elevated by antidepressants in rat brain.

OBJECTIVE: Beta-arrestins play a pivotal role in G protein-coupled receptor desensitization. beta-Arrestins interfere in G protein receptor interaction, thus leading to desensitization of G protein-mediated receptor signaling. G protein receptor signaling and its desensitization were previously implicated in the pathophysiology of mood disorders and in the mechanism of action of antidepressant and mood-stabilizing treatments. The present study aims at quantitatively evaluating beta-arrestin-1 levels in leukocytes of patients with major depression and the effect of antidepressants on beta-arrestin-1 levels in rat brain. METHOD: Beta-arrestin-1 measurements were carried out in cortical, hippocampal, and striatal brain regions of rats chronically intragastrically treated with either imipramine, desipramine, or fluvoxamine. Similar measurements were conducted in mononuclear leukocytes of 36 untreated patients with major depression and 32 healthy volunteer subjects. Beta-arrestin-1 levels were evaluated through immunoblot analyses using monoclonal antibodies to beta-arrestin-1. RESULTS: Beta-arrestin-1 levels were significantly elevated by all three antidepressants in rat cortex and hippocampus, while in the striatum no alterations could be detected. This process became significant within 10 days and took 2-3 weeks to reach maximal increase. Mononuclear leukocytes of patients with depression showed significantly reduced immunoreactive quantities of beta-arrestin-1. The reduction in beta-arrestin-1 levels was significantly correlated with the severity of depressive symptoms. CONCLUSIONS: The findings in the rat study suggest beta-arrestin-1 elevation as a biochemical mechanism for antidepressant-induced receptor down-regulation. The findings in human subjects support the implication of beta-arrestin-1 in the pathophysiology of mood disorders. Beta-arrestin-1 measurements in patients with depression may potentially serve as a biochemical marker for depression.

G protein-coupled receptor kinase/beta-arrestin systems and drugs of abuse: psychostimulant and opiate studies in knockout mice.

G protein-coupled receptors (GPCRs) currently represent pharmaceutical targets for numerous medicinal compounds that are used to treat conditions ranging from blood pressure dysregulation to depression to pain, demonstrating the wide range of functions mediated by this receptor family. GPCR activation is determined not only by the initiation of signaling cascades but also by regulatory mechanisms that control the extent and duration of their signals. The balance of activation and desensitization dictate the ultimate physiological response to both endogenous and exogenous receptor stimuli. Therefore, these mechanisms may play a particularly relevant role during chronic exposure to agonists such as in conditions when drugs are abused. Two major classes of drugs of abuse, opiates and psychostimulants, both use either direct or indirect GPCR signaling mechanisms to mediate their effects. Therefore, the regulation of GPCRs may have bearing on the neuronal adaptations that underlie the reinforcing properties of drugs of abuse.

Regulation of GRK 2 and 6, beta-arrestin-2 and associated proteins in the prefrontal cortex of drug-free and antidepressant drug-treated subjects with major depression.

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK 2, GRK 6, beta-arrestin-2 and associated proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in parallel (immunodensity with specific antibodies) in brains of depressed subjects (drug-free and antidepressant-treated) to investigate the effect of major depression and antidepressant drugs on these receptor regulatory proteins. Specimens of the prefrontal cortex (Brodmann's area 9) were collected from 19 suicide and non-suicide depressed subjects and 13 control subjects. In drug-free (n=9), but not in antidepressant-treated (n=10), depressed subjects an increase in the density of membrane-associated GRK 2 (30%, n=9, P=0.005) was found compared with that in sex-, age-, and PMD-matched controls. Comparison between drug-free and antidepressant-treated depressed subjects showed that GRK 2 was reduced in membrane (39%, n=10, P=0.008) and cytosolic (44%, n=10, P=0.09) preparations after antidepressant drug treatment. In contrast, membrane-associated GRK 6 (drug-free and antidepressant-treated depressed subjects) was found unchanged when compared with that in matched controls. Similarly, the densities of beta-arrestin-2, PP-2A, and Gbeta proteins were not significantly different from those in matched controls. There was a positive correlation between the immunodensities of GRK 2 and beta-arrestin-2 in membrane preparations (r=0.48, n=19, P=0.04), suggesting that both proteins are regulated in a coordinated manner in brains of depressed subjects. The results of this study indicate that major depression is associated with upregulation of brain GRK 2, but not GRK 6, and that antidepressant drug treatment appears to induce downregulation of GRK 2 protein.

Beta -Arrestin 1 down-regulation after insulin treatment is associated with supersensitization of beta 2 adrenergic receptor Galpha s signaling in 3T3-L1 adipocytes.

beta-Arrestin 1 is required for internalization and mitogen-activated protein (MAP) kinase activation by the beta2 adrenergic receptor (beta2AR). Our previous studies have shown that chronic insulin treatment down-regulates cellular beta-arrestin 1 levels, leading to a marked impairment in G protein-coupled receptor and insulin-like growth factor-1 receptor-mediated MAP kinase and mitogenic signaling. In this study, we show that chronic insulin-treated, beta-arrestin 1depleted 3T3-L1 adipocytes display (i) increased isoproterenol-induced cAMP generation (53 +/- 38% at 1.5 min, 25 +/- 19% at 5 min, 63 +/- 14% at 30 min, and 59 +/- 2% at 60 min), a Galpha(s)-associated pathway; (ii) impaired isoproterenol-induced beta2AR internalization (reduced by 98 +/- 4%), which is required for MAP kinase signaling, a Galpha(i)-associated pathway; and (iii) increased beta-arrestin 1 phosphorylation at Ser-412. Taken together, these findings represent a hitherto unknown mechanism (degradation and phosphorylation of beta-arrestin, whereby the activation of the insulin receptor, belonging to the family of receptor tyrosine kinases, causes supersensitization of Galpha(s)-associated signaling and inhibition of Galpha(i)-associated signaling by the beta2AR, a prototypical G protein-coupled receptor.