Effects of ß-Arrestin-Biased Dopamine D2 Receptor Ligands on Schizophrenia-Like Behavior in Hypoglutamatergic Mice.

Current antipsychotic drugs (APDs) show efficacy with positive symptoms, but are limited in treating negative or cognitive features of schizophrenia. Whereas all currently FDA-approved medications target primarily the dopamine D2 receptor (D2R) to inhibit G(i/o)-mediated adenylyl cyclase, a recent study has shown that many APDs affect not only G(i/o)- but they can also influence ß-arrestin- (ßArr)-mediated signaling. The ability of ligands to differentially affect signaling through these pathways is termed functional selectivity. We have developed ligands that are devoid of D2R-mediated G(i/o) protein signaling, but are simultaneously partial agonists for D2R/ßArr interactions. The purpose of this study was to test the effectiveness of UNC9975 or UNC9994 on schizophrenia-like behaviors in phencyclidine-treated or NR1-knockdown hypoglutamatergic mice. We have found the UNC compounds reduce hyperlocomotion in the open field, restore PPI, improve novel object recognition memory, partially normalize social behavior, decrease conditioned avoidance responding, and elicit a much lower level of catalepsy than haloperidol. These preclinical results suggest that exploitation of functional selectivity may provide unique opportunities to develop drugs with fewer side effects, greater therapeutic selectivity, and enhanced efficacy for treating schizophrenia and related conditions than medications that are currently available.

ß-arrestin regulates estradiol membrane-initiated signaling in hypothalamic neurons.

Estradiol (E2) action in the nervous system is the result of both direct nuclear and membrane-initiated signaling (EMS). E2 regulates membrane estrogen receptor-α (ERα) levels through opposing mechanisms of EMS-mediated trafficking and internalization. While ß-arrestin-mediated mERα internalization has been described in the cortex, a role of ß-arrestin in EMS, which underlies multiple physiological processes, remains undefined. In the arcuate nucleus of the hypothalamus (ARH), membrane-initiated E2 signaling modulates lordosis behavior, a measure of female sexually receptivity. To better understand EMS and regulation of ERα membrane levels, we examined the role of ß-arrestin, a molecule associated with internalization following agonist stimulation. In the present study, we used an immortalized neuronal cell line derived from embryonic hypothalamic neurons, the N-38 line, to examine whether ß-arrestins mediate internalization of mERα. ß-arrestin-1 (Arrb1) was found in the ARH and in N-38 neurons. In vitro, E2 increased trafficking and internalization of full-length ERα and ERαΔ4, an alternatively spliced isoform of ERα, which predominates in the membrane. Treatment with E2 also increased phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2) in N-38 neurons. Arrb1 siRNA knockdown prevented E2-induced ERαΔ4 internalization and ERK1/2 phosphorylation. In vivo, microinfusions of Arrb1 antisense oligodeoxynucleotides (ODN) into female rat ARH knocked down Arrb1 and prevented estradiol benzoate-induced lordosis behavior compared with nonsense scrambled ODN (lordosis quotient: 3 ± 2.1 vs. 85.0 ± 6.0; p < 0.0001). These results indicate a role for Arrb1 in both EMS and internalization of mERα, which are required for the E2-induction of female sexual receptivity.

Cell-based assays and animal models for GPCR drug screening.

The family of G protein-coupled receptors (GPCRs) remains a central focus of basic pharmacology and drug discovery efforts. Convenient methods to assess the efficacy of potentially therapeutic reagents for GPCRs are strongly required for high-throughput screening (HTS) assay. We recently developed a rapid, sensitive, and quantitative method for detecting potential chemicals that act on GPCRs using split luciferase complementation. In principle, this is based on the detection of interactions of GPCR with ß-arrestin, which translocates to the activated GPCRs. This method can facilitate the construction of HTS systems in a multi-well plate format. Particularly, the method is compatible with single-cell imaging and animal models and even deeper tissues such as organs, because of its high sensitivity, suggesting that promising candidates from HTS assay can be moved easily to the next phase for additional analysis. This system can contribute to the effective evaluation of potentially therapeutic reagents and expedite the development of new drugs for GPCRs.

Intervention effects of QRZSLXF, a Chinese medicinal herb recipe, on the DOR-ß-arrestin1-Bcl2 signal transduction pathway in a rat model of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingre Zaoshi Liangxue Fang (QRZSLXF) is a Chinese medicinal herb recipe that is commonly prescribed for the treatment of ulcerative colitis. It includes 5 quality assured herbs: Sophora flavescens Aiton., Baphicacanthus cusia (Nees) Bremek., Bletilla striata Rchb.f., Glycyrrhiza uralensis Fisch. and Coptis chinensis Franch. The main phytochemical ingredient of QRZSLXF includes ammothamnine, sophocarpidine, liquiritin, berberine and indirubin. QRZSLXF has been clinically proven for use in the treatment of ulcerative colitis for over twenty years. In the past ten years, research has confirmed the therapeutic effect of QRZSLXF in ulcerative colitis and partially revealed its mechanism of action. Here, we further reveal the therapeutic mechanism of QRZSLXF in ulcerative colitis. To investigate the role of the DOR-ß-arrestin1-Bcl-2 signal transduction pathway in ulcerative colitis and to determine the effects of QRZSLXF on this signal transduction pathway. MATERIALS AND METHODS: Eighty-four Sprague-Dawley rats were randomly divided into six groups: normal control group, model group, mesalazine group, and QRZSLXF high-dose, medium-dose group and low-dose groups (n=14). Experimental colitis was induced by trinitrobenzenesulfonic acid (TNBS) in each group, except the normal control group. After modeling, bloody stool, mental state and diarrhea were observed and recorded. Two rats were randomly selected from the model groups adfnd sacrificed on day 3 to observe pathological changes in the colon tissue by microscopy. The rats in the QRZSLXF-treated groups received intramuscular injections of different concentrations of QRZSLXF for 15 days. The rats in the mesalazine group were treated with mesalazine solution (0.5 g/kg/day) by gastric lavage for 15 days. The rats in the normal control group and the model group were treated with 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the remaining rats were sacrificed and their colon tissues were used to detect the mRNA and protein expressions of DOR, ß-arrestin1 and Bcl-2 by Real-time PCR and immunohistochemistry, respectively. Histological changes in the colon tissues were also examined. RESULTS AND CONCLUSIONS: The expressions of DOR, ß-arrestin1 and Bcl-2 were significantly different among the four groups. The expressions of DOR, ß-arrestin1 and Bcl-2 protein and mRNA were significantly increased in the model group compared with the other groups (P<0.05). In contrast to the model group, the expressions of DOR, ß-arrestin1 and Bcl-2 were significantly decreased in the mesalazine group and the groups that received different doses of QRZSLXF (P<0.05), and there were no statistically significant differences among the mesalazine and QRZSLXF-treated groups (P>0.05). This study indicates that the DOR-beta-arrestin1-Bcl-2 signal transduction pathway may participate in the pathologic course of ulcerative colitis. Moreover, QRZSLXF could attenuate ulcerative colitis by regulating the DOR-ß-arrestin1-Bcl-2 signal transduction pathway.

Effect of Tianqi antitremor granules on behavioral manifestations and expression of G protein-coupled receptor kinase 6 and ß-arrestin1 in levodopa-induced dyskinesia in a rat model of Parkinson's disease.

BACKGROUND: Recent studies have shown that expression of G protein-coupled receptor kinase 6 (GRK6) and ß-arrestin1 in the striatum is closely associated with hyperactive dopamine receptors in rats with levodopa-induced dyskinesia (LID). Our research group has shown that Tianqi antitremor granules have a significant effect on the motor complications of Parkinson's disease (PD). However, whether Tianqi antitremor granules have an effect on the behavioral manifestations and expression of GRK6 and ß-arrestin1 in rats with LID is unknown. METHODS: Rats with PD received twice daily intraperitoneal injections of levodopa for 4 weeks to induce dyskinesia. Rats with LID were randomly divided into five groups: an LID-control group, an LID group, a levodopa plus Tianqi antitremor granules as traditional Chinese medicine (TCM)-low group, a levodopa plus TCM-medium group, and levodopa plus TCM-high group. Peak intensity of rotations was measured. GRK6 and ß-arrestin1 expression in the striatum of the dyskinetic rats was observed by immunohistochemistry and Western blotting. RESULTS: Pulsatile treatment with levodopa induced abnormal involuntary movements in rats with PD similar to LID in patients with PD. We found that repeated levodopa administration increased peak rotations in dyskinetic rats. However, peak rotations were decreased in rats given levodopa plus the different doses of Tianqi antitremor granules. In accordance with changed behavior, GRK6 and ß-arrestin1 expression was decreased in rats with PD and was persistently low in rats with LID, but this decrease was prevented by coadministration of levodopa and Tianqi antitremor granules. CONCLUSION: Tianqi antitremor granules ameliorated levodopa-induced dyskinetic behavior, reversed the decrease in GRK6 and ß-arrestin1 expression, and acted as a useful adjunctive medicine for the treatment of LID.

Effects of Chinese Fructus Mume formula and its separated prescription extract on insulin resistance in type 2 diabetic rats.

The effect of Fructus Mume formula and its separated prescription extract on insulin resistance in type 2 diabetic rats was investigated. The rat model of type 2 diabetes was established by feeding on a high-fat diet for 8 weeks and by subsequently intravenous injection of small doses of streptozotocin. Rats in treatment groups, including the Fructus Mume formula treatment group (FM), the cold property herbs of Fructus Mume formula treatment group (CFM), the warm property herbs of Fructus Mume formula treatment group (WFM), were administrated with Fructus Mume formula and its separated prescription extract by gavage, while the rats in diabetic model group (DM) and metformin group (MET) were given by gavage with normal saline and metformin correspondingly. The body weight before and after treatment was measured, and the oral glucose tolerance test (OGTT) and the insulin release test (IRT) were performed. The homeostasis model assessment-insulin resistance index (HOMA-IR) was calculated. The protein and mRNA expression levels of Insr, ß-arrestin-2, Irs-1 and Glut-4 in the liver, skeletal muscle and fat tissues were detected by using Western blotting and RT-PCR respectively. The results demonstrated that, as compared with DM group, OGTT, IRT (0 h, 1 h) levels and HOMR-IR in treatment groups were all reduced, meanwhile their protein and mRNA expression levels of Insr, Irs-1 and Glut-4 in the liver, skeletal muscle and fat tissues were obviously increased, and their protein and mRNA expression levels of ß-arrestin-2 in the liver and skeletal muscle tissues were also markedly increased. It was suggested that the Fructus Mume formula and its separated prescription extracts could effectively improve insulin resistance in type 2 diabetic rats, which might be related to the up-regulated expression of Insr, Irs-1 and Glut-4 in the liver, skeletal muscle and fat tissues, and ß-arrestin-2 in the liver and skeletal muscle tissues.

Intrahepatic expression of genes related to metabotropic receptors in chronic hepatitis.

AIM: To screen for genes related to metabotropic receptors that might be involved in the development of chronic hepatitis. METHODS: Assessment of 20 genes associated with metabotropic receptors was performed in liver specimens obtained by punch biopsy from 12 patients with autoimmune and chronic hepatitis type B and C. For this purpose, a microarray with low integrity grade and with oligonucleotide DNA probes complementary to target transcripts was used. Evaluation of gene expression was performed in relation to transcript level, correlation between samples and grouping of clinical parameters used in chronic hepatitis assessment. Clinical markers of chronic hepatitis included alanine and aspartate aminotransferase, γ-glutamyltranspeptidase, alkaline phosphatase and cholinesterase activity, levels of iron ions, total cholesterol, triglycerides, albumin, glucose, hemoglobin, platelets, histological analysis of inflammatory and necrotic status, fibrosis according to METAVIR score, steatosis, as well as anthropometric body mass index, waist/hip index, percentage of adipose tissue and liver size in ultrasound examination. Gender, age, concomitant diseases and drugs were also taken into account. Validation of oligonucleotide microarray gene expression results was done with the use of quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The highest (0.002 < P < 0.046) expression among genes encoding main components of metabotropic receptor pathways, such as the α subunit of G-coupled protein, phosphoinositol-dependent protein kinase or arrestin was comparable to that of angiotensinogen synthesized in the liver. Carcinogenesis suppressor genes, such as chemokine ligand 4, transcription factor early growth response protein 1 and lysophosphatidic acid receptor, were characterized by the lowest expression (0.002 < P < 0.046), while the factor potentially triggering hepatic cancer, transcription factor JUN-B, had a 20-fold higher expression. The correlation between expression of genes of protein kinases PDPK1, phosphoinositide 3-kinase and protein kinase A (Spearman's coefficient range: 0.762-0.769) confirmed a functional link between these enzymes. Gender (P = 0.0046) and inflammation severity, measured by alanine aminotransferase activity (P = 0.035), were characterized by diverse metabotropic receptor gene expression patterns. The Pearson's coefficient ranging from -0.35 to 0.99 from the results of qRT-PCR and microarray indicated that qRT-PCR had certain limitations as a validation tool for oligonucleotide microarray studies. CONCLUSION: A microarray-based analysis of hepatocyte metabotropic G-protein-related gene expression can reveal the molecular basis of chronic hepatitis.

Pollen Typhae total flavone improves insulin-induced glucose uptake through the ß-arrestin-2-mediated signaling in C2C12 myotubes.

Defects in insulin-stimulated glucose uptake in skeletal muscle result from the dysfunction of insulin signaling including the phosphatidylinositol-3 kinase (PI3K) pathway and a novel ß-arrestin-2-mediated signaling, which leads to insulin resistance (IR). Pollen Typhae, a Chinese herb, has been used for thousands of years in traditional Chinese medicine, and has the potential to inhibit the development of IR. We have previously reported that Pollen Typhae total flavone (PTF), the extract from Pollen Typhae, ameliorates high-glucose- and high-insulin-induced impairment of glucose uptake in 3T3-L1 adipocytes, but the mechanisms remain unclear. The objective of this study was to investigate the effects of PTF on glucose uptake, and to explore the underlying mechanisms in C2C12 myotubes. PTF improved insulin-stimulated glucose uptake in a dose- and time-dependent manner in C2C12 myotubes, and prevented palmitate-induced IR. Furthermore, PTF enhanced the basal gene expression of Src and Akt2, elevated the protein expression of ß-arrestin-2, Src and Akt, increased the phosphorylation of insulin receptor-ß at Tyr1150/1151 and Akt at Thr308/Ser473 in an insulin-dependent manner, but had no effects on the protein expression of PI3K-p85 or the activity of PI3K. Inhibition of Src but not PI3K restrained PTF-induced phosphorylation of Akt and glucose uptake. Our findings indicate that PTF improves insulin-induced glucose uptake via the ß-arrestin-2-mediated signaling in C2C12 myotubes.

A high-throughput screening system for G-protein-coupled receptors using ß-lactamase enzyme complementation technology.

AIM: to establish a system for monitoring the activation of G-protein-coupled receptors (GPCRs) using ß-lactamase enzyme fragment complementation (EFC) technology. METHODS: two inactive ß-lactamase deletion fragments, bla(a) and bla(b), were fused to ß-arrestin and GPCR, respectively. A stable cell line named HEK/293-ß2a2, which expressed two fusion proteins, GPCR/bla(b) and ß-arrestin2/bla(a), was generated under antibiotic selection. A natural compound library of high performance liquid chromatography (HPLC)-fractionated samples from the ethanol extracts of Chinese medicinal herbs was used for high-throughput screening (HTS) of ß2-adrenoceptor (ß2AR) agonists against the cell line HEK/293-ß2a2. The interested hits were validated by the measurement of second-messenger cyclic adenosine monophosphate (cAMP) production. RESULTS: the stable cell line HEK/293-ß2a2 responded to ß2AR agonist/antagonist in a dose-dependent manner. The EC(50) value obtained for isoproterenol was 15.5 nmol/L, and the IC(50) value obtained for propranolol was 51.9 nmol/L. Furthermore, HTS was performed to identify ß2AR agonists from the natural compound library we established. The Z' factor value was determined to be 0.68. Three hits were identified from primary screening and found to be as potent as isoproterenol in a camp assay. CONCLUSION: a cell-based high-throughput functional assay was established to directly monitor the activation of GPCRs based on the interaction between agonist-activated GPCR and ß-arrestin using ß-lactamase EFC technology, which can be used to search for leads in the natural compound library.

GPR54 regulates ERK1/2 activity and hypothalamic gene expression in a Gα(q/11) and ß-arrestin-dependent manner.

G protein-coupled receptor 54 (GPR54) is a G(q/11)-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP(2) hydrolysis, Ca(2+) mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled G(q/11) and ß-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using G(q/11) and ß-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the G(q/11) and ß-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while ß-arrestin-2 potentiates GPR54 signaling to ERK, ß-arrestin-1 inhibits it. Our data also revealed that diminished ß-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, ß-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the G(q/11) pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation.

Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression.

Understanding the physiopathology of affective disorders and their treatment relies on the availability of experimental models that accurately mimic aspects of the disease. Here we describe a mouse model of an anxiety/depressive-like state induced by chronic corticosterone treatment. Furthermore, chronic antidepressant treatment reversed the behavioral dysfunctions and the inhibition of hippocampal neurogenesis induced by corticosterone treatment. In corticosterone-treated mice where hippocampal neurogenesis is abolished by X-irradiation, the efficacy of fluoxetine is blocked in some, but not all, behavioral paradigms, suggesting both neurogenesis-dependent and -independent mechanisms of antidepressant action. Finally, we identified a number of candidate genes, the expression of which is decreased by chronic corticosterone and normalized by chronic fluoxetine treatment selectively in the hypothalamus. Importantly, mice deficient in one of these genes, beta-arrestin 2, displayed a reduced response to fluoxetine in multiple tasks, suggesting that beta-arrestin signaling is necessary for the antidepressant effects of fluoxetine.

Development of a BRET2 screening assay using beta-arrestin 2 mutants.

This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET(2) signal. The beta-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET(2)-based agonist and antagonist screening assays.

Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology.

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.

Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor is mediated by a serine residue close to the COOH terminus.

The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect internalization of the receptor to an arrestin- and clathrin-independent pathway.

Synergistic regulation of m2 muscarinic acetylcholine receptor desensitization and sequestration by G protein-coupled receptor kinase-2 and beta-arrestin-1.

The m2 muscarinic acetylcholine receptor (m2 mAChR) belongs to the superfamily of G protein-coupled receptors and is regulated by many processes that attenuate signaling following prolonged stimulation by agonist. We used a heterologous expression system to examine the ability of G protein-coupled receptor kinase-2 (GRK2) and beta-arrestin-1 to regulate the phosphorylation state and to promote desensitization and sequestration of the m2 mAChR. Treatment of JEG-3 cells transiently expressing the m2 mAChR with a muscarinic agonist induced an approximately 4- or 8-fold increase in receptor phosphorylation in the absence or presence of cotransfected GRK2, respectively, compared with untreated cells transfected with receptor alone. Using the expression of a cAMP-regulated reporter gene to measure receptor function, we found that transiently transfected m2 mAChRs underwent functional desensitization following exposure to agonist. Transfected GRK2 enhanced agonist-induced functional desensitization in a manner that was synergistically enhanced by cotransfection of beta-arrestin-1, which had no effect on m2 mAChR function when coexpressed in the absence of GRK2. Finally, GRK2 and beta-arrestin-1 synergistically enhanced both the rate and extent of agonist-induced m2 mAChR sequestration. These results are the first to demonstrate that agonist-induced desensitization and sequestration of the m2 mAChR in the intact cell can be enhanced by the presence of GRK2 and beta-arrestin-1 and show that these molecules have multiple actions on the m2 mAChR.