Analysis of Arrestin Recruitment to Chemokine Receptors by Bioluminescence Resonance Energy Transfer.

Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.

The emerging roles of ß-arrestins in fibrotic diseases.

ß-Arrestins and ß-arrestin2 are important adaptor proteins and signal transduction proteins that are mainly involved in the desensitization and internalization of G-protein-coupled receptors. Fibrosis is characterized by accumulation of excess extracellular matrix (ECM) molecules caused by chronic tissue injury. If highly progressive, the fibrotic process leads to organ malfunction and, eventually, death. The incurable lung fibrosis, renal fibrosis and liver fibrosis are among the most common fibrotic diseases. Recent studies show that ß-arrestins can activate signaling cascades independent of G-protein activation and scaffold many intracellular signaling networks by diverse types of signaling pathways, including the Hedgehog, Wnt, Notch and transforming growth factor-ß pathways, as well as downstream kinases such as MAPK and PI3K. These signaling pathways are involved in the pathological process of fibrosis and fibrotic diseases. This ß-arrestin-mediated regulation not only affects cell growth and apoptosis, but also the deposition of ECM, activation of inflammatory response and development of fibrotic diseases. In this review, we survey the involvement of ß-arrestins in various signaling pathways and highlight different aspects of their regulation of fibrosis. We also discuss the important roles of ß-arrestins in the process of fibrotic diseases by regulating the inflammation and deposit of ECM. It is becoming more evident that targeting ß-arrestins may offer therapeutic potential for the treatment of fibrotic diseases.

Expression of vasoactive proteins in gastric antral mucosa reflects vascular dysfunction in patients with cirrhosis and portal hypertension.

BACKGROUND & AIMS: Patients with cirrhosis display hypocontractility of splanchnic vessels because of dysregulation of vasoactive proteins, such as decreased effect of RhoA/ROCK and increased activity of ß-Arrestin-2 and eNOS. However, it is unknown whether the dysregulation of vasoactive proteins is displayed in other vessels. We investigated whether expression of vasoactive proteins can be evaluated in gastric mucosa vessels. METHODS: Biopsies from the gastric mucosa of 111 patients with cirrhosis were collected at three different centres and from 13 controls. Forty-nine patients had received TIPS. Portal pressure gradient was measured in 49 patients with TIPS and in 16 patients without TIPS. Biopsies from the antrum were conserved in formaldehyde for immunohistochemistry or shock-frozen for PCR and Western blot. RESULTS: The mucosal transcription of vascular markers (αSMA, CD31) was higher in cirrhotic patients than controls, which was confirmed by immunohistochemistry. On average, relative mucosal levels of RhoA and ROCK were lower, while ß-Arrestin-2 levels were higher in cirrhotic patients compared to controls. Transcriptional levels of eNOS increased with presence of ascites and grade of oesophageal varices. Patients with TIPS showed less pronounced markers of vascular dysfunction in gastric mucosa. CONCLUSION: This is the first evidence that the expression of vasoactive proteins in mucosa from the gastric antrum of patients with cirrhosis reflects their vascular dysfunction and possibly changes after therapeutic interventions.

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C-C chemokine receptor 5 with RANTES/CCL5 analogues.

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues.

A simple method to generate stable cell lines for the analysis of transient protein-protein interactions.

Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energy transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins.

Multiparametric homogeneous method for identification of ligand binding to G protein-coupled receptors: receptor-ligand binding and ß-arrestin assay.

Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to ß(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of ß(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.

ß-Arrestins and G protein-coupled receptor trafficking.

Arrestins are adaptor proteins that function to regulate G protein-coupled receptor (GPCR) signaling and trafficking. There are four mammalian members of the arrestin family, two visual and two nonvisual. The visual arrestins (arrestin-1 and arrestin-4) are localized in rod and cone cells, respectively, and function to quench phototransduction by inhibiting receptor/G protein coupling. The nonvisual arrestins (ß-arrestin1 and ß-arrestin2, a.k.a. arrestin-2 and arrestin-3) are ubiquitously expressed and function to inhibit GPCR/G protein coupling and promote GPCR trafficking and arrestin-mediated signaling. Arrestin-mediated endocytosis of GPCRs requires the coordinated interaction of ß-arrestins with clathrin, adaptor protein 2, and phosphoinositides such as PIP(2)/PIP(3). These interactions are facilitated by a conformational change in ß-arrestin that is thought to occur upon binding to a phosphorylated activated GPCR. In this chapter, we provide an overview of the reagents and techniques used to study ß-arrestin-mediated receptor trafficking.

Drosulfakinin activates CCKLR-17D1 and promotes larval locomotion and escape response in Drosophila.

Neuropeptides are ubiquitous in both mammals and invertebrates and play essential roles in regulation and modulation of many developmental and physiological processes through activation of G-protein-coupled-receptors (GPCRs). However, the mechanisms by which many of the neuropeptides regulate specific neural function and behaviors remain undefined. Here we investigate the functions of Drosulfakinin (DSK), the Drosophila homolog of vertebrate neuropeptide cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system. We provide biochemical evidence that sulfated DSK-1 and DSK-2 activate the CCKLR-17D1 receptor in a cell culture assay. We further examine the role of DSK and CCKLR-17D1 in the regulation of larval locomotion, both in a semi-intact larval preparation and in intact larvae under intense light exposure. Our results suggest that DSK/CCKLR-17D1 signaling promote larval body wall muscle contraction and is necessary for mediating locomotor behavior in stress-induced escape response.

Visualization and quantitative analysis of G protein-coupled receptor-ß-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation.

Methods used to assess the efficacy of potentially therapeutic reagents for G protein-coupled receptors (GPCRs) have been developed. Previously, we demonstrated sensitive detection of the interaction of GPCRs and ß-arrestin2 (ARRB2) using 96-well microtiter plates and a bioluminescence microscope based on split click beetle luciferase complementation. Herein, using firefly luciferase emitting longer wavelength light, we demonstrate quantitative analysis of the interaction of ß2-adrenergic receptor (ADRB2), a kind of GPCR, and ARRB2 in a 96-well plate assay with single-cell imaging. Additionally, we showed bioluminescence in vivo imaging of the ADRB2-ARRB2 interaction in two systems: cell implantation and hydrodynamic tail vein (HTV) methods. Specifically, in the HTV method, the luminescence signal from the liver upon stimulation of an agonist for ADRB2 was obtained in the intact systems of mice. The results demonstrate that this method enables noninvasive screening of the efficacy of chemicals at the specific organ in in vivo testing. This in vivo system can contribute to effective evaluation in pharmacokinetics and pharmacodynamics and expedite the development of new drugs for GPCRs.

The role of ß-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract.

ß-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of ß-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 µM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from ß-arrestin2 knockout mice. ß-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of ß-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 µM) exposure. Naloxone prevented the decrease in ß-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in ß-arrestin2 occurred. We conclude that a decrease in ß-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

A selective cysteinyl leukotriene receptor 2 antagonist blocks myocardial ischemia/reperfusion injury and vascular permeability in mice.

Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed ß-galactosidase-ß-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be ∼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 µM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.

Study of G protein-coupled receptor/ß-arrestin interactions within endosomes using FRAP.

ß-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of ß-arrestin/receptor interactions and how these complexes, and complexes with other regulatory proteins, are controlled in cells. Here, we use yellow fluorescent protein (YFP)-tagged ß-arrestin 2 and a fluorescence recovery after photobleaching (FRAP) imaging approach to probe the real-time interaction of ß-arrestin with a GPCR, the bradykinin type 2 receptor (B2R). We provide a detailed protocol to assess the avidity of ß-arrestin2-YFP for B2R within endosomes in HEK293 cells. ß-arrestin2-YFP associated with internalized receptors is photobleached with intense light, and fluorescence recovery due to the entry of nonbleached ß-arrestin2-YFP is monitored over time as a measure of the rate exchange of ß-arrestin2-YFP within the endosome. This approach can be extended to other GPCR/ß-arrestin complexes and their putative regulators to provide information about the kinetics of similar protein-protein interactions in cells. Moreover, these techniques should provide insight into the role of ß-arrestins in the intracellular trafficking and signaling of GPCRs.

Morphine induces µ opioid receptor endocytosis in guinea pig enteric neurons following prolonged receptor activation.

BACKGROUND & AIMS: The µ opioid receptor (µOR) undergoes rapid endocytosis after acute stimulation with opioids and most opiates, but not with morphine. We investigated whether prolonged activation of µOR affects morphine's ability to induce receptor endocytosis in enteric neurons. METHODS: We compared the effects of morphine, a poor µOR-internalizing opiate, and (D-Ala2,MePhe4,Gly-ol5) enkephalin (DAMGO), a potent µOR-internalizing agonist, on µOR trafficking in enteric neurons and on the expression of dynamin and ß-arrestin immunoreactivity in the ileum of guinea pigs rendered tolerant by chronic administration of morphine. RESULTS: Morphine (100 µmol/L) strongly induced endocytosis of µOR in tolerant but not naive neurons (55.7% ± 9.3% vs 24.2% ± 7.3%; P < .001) whereas DAMGO (10 µmol/L) strongly induced internalization of µOR in neurons from tolerant and naive animals (63.6% ± 8.4% and 66.5% ± 3.6%). Morphine- or DAMGO-induced µOR endocytosis resulted from direct interactions between the ligand and the µOR because endocytosis was not affected by tetrodotoxin, a blocker of endogenous neurotransmitter release. Ligand-induced µOR internalization was inhibited by pretreatment with the dynamin inhibitor, dynasore. Chronic morphine administration resulted in a significant increase and translocation of dynamin immunoreactivity from the intracellular pool to the plasma membrane, but did not affect ß-arrestin immunoreactivity. CONCLUSIONS: Chronic activation of µORs increases the ability of morphine to induce µOR endocytosis in enteric neurons, which depends on the level and cellular localization of dynamin, a regulatory protein that has an important role in receptor-mediated signal transduction in cells.

Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores.

The fusion of fluorescent proteins to proteins of interest has greatly advanced fluorescence microscopy, but is often limited by their large size. Here, we report site-specific, orthogonal labeling of two cellular proteins in intact cells with two small fluorescent dyes: fluorescein arsenical hairpin binder, FlAsH, and its red analogue, ReAsH, which bind to tetracysteine motifs. Development of a sequential labeling method to two different motifs, CCPGCC and FLNCCPGCCMEP, allowed site-specific labeling with FlAsH and ReAsH, respectively. Using the cell surface receptor for parathyroid hormone and its cytosolic binding protein, beta-arrestin2, we show their selective visualization in intact cells and analyze their interaction by colocalization and fluorescence resonance energy transfer (FRET). We propose that this method may be widely applied to label intracellular proteins and to study their interactions in intact cells with minimal disturbance of their function.

Improved donor/acceptor BRET couples for monitoring beta-arrestin recruitment to G protein-coupled receptors.

We report highly sensitive bioluminescence resonance energy transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein, the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced beta-arrestin (betaARR) intramolecular rearrangements and recruitment to G protein-coupled receptors. We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing betaARR assays and offer new options for the development of future BRET assays.

ß-arrestins attenuate p38-mediated endosome to Golgi transport.

Shiga toxin (Stx) is after endocytosis transported via early endosomes to the Golgi apparatus and endoplasmic reticulum. It is then translocated to the cytosol where it exerts its toxic effect. We recently reported that p38 is required for endosome to Golgi transport of Stx. In the present study, we investigated whether ß-arrestins are effectors of this pathway. ß-arrestin knockdown led to enhanced Stx transport. A similar phenotype was achieved upon p38 activation. We demonstrate that p38 and ß-arrestin act on the same pathway. ß-arrestin colocalized with internalized Stx and, interestingly, was recruited to endosomes upon p38 activation. After Stx treatment, p38 and ß-arrestin formed a transient complex. From these data we propose that ß-arrestin negatively regulates Stx transport via an interaction with activated p38 and attenuation of its signalling. Interestingly, also mannose 6-phosphate receptor transport was regulated by p38 and ß-arrestin. ß-arrestins therefore seem to regulate an endosome to Golgi pathway used by multiple cargo proteins.

Characterizing cannabinoid CB2 receptor ligands using DiscoveRx PathHunter beta-arrestin assay.

The authors have characterized a set of cannabinoid CB(2) receptor ligands, including triaryl bis sulfone inverse agonists, in a cell-based receptor/beta-arrestin interaction assay (DiscoveRx PathHunter). The results were compared with results using a competitive ligand binding assay, and with effects on forskolin-stimulated cAMP levels (PerkinElmer LANCE). The authors show good correlation between the 3 assay systems tested, with the beta-arrestin protein complementation assay exhibiting a more robust signal than the cAMP assay for cannabinoid CB(2) agonists. Further assay validation shows that DiscoveRx PathHunter HEK293 CB(2) beta-arrestin assay can be carried out from cryopreserved cell suspensions, eliminating variations caused by the need for multiple cell pools during live cell screening campaigns. These results, and the authors' results evaluating a test set of random library compounds, validate the use of ligand-induced interaction between the human cannabinoid CB(2) receptor and beta-arrestin as an appropriate and valuable screening platform for compounds specific for the cannabinoid CB(2) receptor.

Functional characterization of kurtz, a Drosophila non-visual arrestin, reveals conservation of GPCR desensitization mechanisms.

The arrestins are a family of molecules that terminate signaling from many different G protein-coupled receptors, by inhibiting the association between receptor and downstream effectors. We recently employed a human betaarrestin2-GFP fusion protein to explore the dynamics of different neuropeptide receptors in Drosophila and have previously used a betaarrestin translocation assay to identify ligands at orphan receptors. Here, we report that the Drosophila arrestin encoded by kurtz functions in a similar fashion and can be employed to investigate GPCR-arrestin associations. Specifically, a GFP-krz fusion protein, upon co-expression with various Drosophila peptide receptors, an amine receptor, and a mammalian peptide receptor translocates to the plasma membrane in specific response to ligand application. This molecular phenotype is exhibited in a mammalian cell line as well as in a Drosophila cell line. Notably, the details of receptor-arrestin associations in terms of endocytotic patterns are functionally conserved between the mammalian arrestins and kurtz. Furthermore, we report that kurtz mutants exhibit hypersensitivity to osmotic stress, implicating GPCR desensitization as an important feature of the endocrine events that shape this stress response.

Expression of G protein-coupled receptor kinase 4 is associated with breast cancer tumourigenesis.

G-protein-coupled receptor kinases (GRKs) comprise a family of seven mammalian serine/threonine protein kinases that phosphorylate and regulate agonist-bound, activated, G-protein-coupled receptors (GPCRs). GRKs and beta-arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization. Here we show that GRK4 isoforms are expressed in human breast cancer but not in normal epithelia. In addition, GRK4-over-expressing cells activated the mitogen-activated protein kinase (MAPK) mediated by ERK 1/2 and JNK phosphorylation in breast cancer-derived cell lines. Furthermore, suppression of beta-arrestins decreased GRK4-stimulated ERK 1/2 or JNK phosphorylations. These data indicate that high-level expression of GRK4 may activate MAPK signalling pathways mediated by beta-arrestins in breast cancer cells, suggesting that GRK4 may be implicated in breast cancer carcinogenesis.

High throughput screening for orphan and liganded GPCRs.

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. The GPCR research platform implemented at Amgen is used as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. The strengths, weaknesses, and applications of assay technologies applicable to G alpha s, G alpha i and G alpha q-coupled receptors are described and their relative merits evaluated. Particular consideration is made of the role and practice of "de-orphaning" and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high throughput hit characterization and prioritization is also discussed extensively.