Targeting arrestin interactions with its partners for therapeutic purposes.

Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds of G protein-coupled receptors, certain receptors of other types, and numerous non-receptor partners. Arrestins have no enzymatic activity and work by interacting with other proteins, often assembling multi-protein signaling complexes. Arrestin binding to every partner affects cell signaling, including pathways regulating cell survival, proliferation, and death. Thus, targeting individual arrestin interactions has therapeutic potential. This requires precise identification of protein-protein interaction sites of both participants and the choice of the side of each interaction which would be most advantageous to target. The interfaces involved in each interaction can be disrupted by small molecule therapeutics, as well as by carefully selected peptides of the other partner that do not participate in the interactions that should not be targeted.

Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.

G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca2+ in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins.

Substance P enhances tissue factor release from granulocyte-macrophage colony-stimulating factor-dependent macrophages via the p22phox/ß-arrestin 2/Rho A signaling pathway.

Granulocyte-macrophage colony stimulating factor (GM-CSF) induces procoagulant activity of macrophages. Tissue factor (TF) is a membrane-bound glycoprotein and substance P (SP) is a pro-inflammatory neuropeptide involved in the formation of membrane blebs. This study investigated the role of SP in TF release by GM-CSF-dependent macrophages. SP significantly decreased TF levels in whole-cell lysates of GM-CSF-dependent macrophages. TF was detected in the culture supernatant by enzyme-linked immunosorbent assay after stimulation of macrophages by SP. Aprepitant (an SP/neurokinin 1 receptor antagonist) reduced TF release from macrophages stimulated with SP. Pretreatment of macrophages with a radical scavenger(pyrrolidinedithiocarbamate) also limited the decrease of TF in whole-cell lysates after stimulation with SP. A protein kinase C inhibitor (rottlerin) partially blocked this macrophage response to SP, while it was significantly inhibited by a ROCK inhibitor (Y-27632) or a dynamin inhibitor (dinasore). An Akt inhibitor (perifosine) also partially blocked this response. Furthermore, siRNA targeting p22phox, ß-arrestin 2, or Rho A, blunted the release of TF from macrophages stimulated with SP. In other experiments, visceral adipocytes derived from cryopreserved preadipocytes were found to produce SP. In conclusion, SP enhances the release of TF from macrophages via the p22phox/ß-arrestin 2/Rho A signaling pathway.

ß-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1.

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic ß cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via ß-arrestins. Further, G protein- and ß-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses ("biased agonism"). Whether GPR40 engages ß-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-ß-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting ß-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of ß-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a ß-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.

Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through ß-arrestin-2 in THP-1 cell line.

The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a ß2-adrenergic receptor (ß2-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of ß2-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and ß-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1ß (IL-1ß) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of ß-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1ß (IL-1ß) release, thus ß-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1ß release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the ß2-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1ß release via ß-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases.

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells.

The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with ß-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, ß-arrestin2, clathrin and ubiquitin-dependent signaling pathway.

Bradykinin-activated contractile signalling pathways in human myometrial cells are differentially regulated by arrestin proteins.

Bradykinin is associated with infections and inflammation, which given the strong correlation between uterine infection and preterm labour may imply that it could play a role in this process. Therefore, we investigated bradykinin signalling, and the roles that arrestin proteins play in their regulation in human myometrial cells. Bradykinin induced rapid, transient intracellular Ca(2+) increases that were inhibited following B2 receptor (B2R) antagonism. Arrestin2 or arrestin3 depletion enhanced and prolonged bradykinin-stimulated Ca(2+) responses, and attenuated B2R desensitisation. Knockdown of either arrestin enhanced B2R-stimulated ERK1/2 signals. Moreover, depletion of either arrestin elevated peak-phase p38-MAPK signalling, yet only arrestin3 depletion prolonged B2R-induced p38-MAPK signals. Arrestin2-knockdown augmented bradykinin-induced cell movement. Bradykinin stimulates pro-contractile signalling mechanisms in human myometrial cells and arrestin proteins play key roles in their regulation. Our data suggest bradykinin not only acts as an utertonin, but may also have the potential to enhance the contractile environment of the uterus.

Absence of a stable secondary structure is not a limitation for photoswitchable inhibitors of ß-arrestin/ß-Adaptin 2 protein-protein interaction.

Many protein-protein interactions (PPIs) are mediated by short, often helical, linear peptides. Molecules mimicking these peptides have been used to inhibit their PPIs. Recently, photoswitchable peptides with little secondary structure have been developed as modulators of clathrin-mediated endocytosis. Here we perform a systematic analysis of a series of azobenzene-crosslinked peptides based on a ß-arrestin P-long 20-mer peptide (BAP-long) sequence to assess the relevance of secondary structure in their interaction with ß-adaptin 2 and to identify the design requirements for photoswitchable inhibitors of PPI (PIPPIs). We observe that flexible structures show a greater inhibitory capacity and enhanced photoswitching ability and that the absence of helical structures in free inhibitor peptide is not a limitation for PIPPI candidates. Therefore, our PIPPIs expand the field of potential inhibitors of PPIs to the wide group of flexible peptides, and we argue against using a stable secondary structure as a sole criterion when designing PIPPI candidates.

Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor.

We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr-EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with ß-arrestins (Arrbs), the small GTPase Rab5, importin-ß (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.

Nicotinic acetylcholine receptors induce c-Kit ligand/Stem Cell Factor and promote stemness in an ARRB1/ ß-arrestin-1 dependent manner in NSCLC.

Lung cancer remains the leading cause of cancer-related deaths worldwide. ß-arrestin-1 (ARRB1), a scaffolding protein involved in the desensitization of signals arising from activated G-protein-coupled receptors (GPCRs), has been shown to play a role in invasion and proliferation of cancer cells, including nicotine-induced proliferation of human non-small cell lung cancers (NSCLCs). In this study, we identified genes that are differentially regulated by nicotine in an ARRB1/ß-arrestin-1 dependent manner in NSCLC cells by microarray analysis. Among the identified genes, SCF (Stem cell factor) strongly differentiated smokers from non-smokers in the Director's Challenge Set expression data and its high expression correlated with poor prognosis. SCF, a major cytokine is the ligand for the c-Kit proto-oncogene and was found to be over expressed in human lung adenocarcinomas, but not squamous cell carcinomas. Data presented here show that transcription factor E2F1 can induce SCF expression at the transcriptional level and depletion of E2F1 or ARRB1/ß-arrestin-1 could not promote self-renewal of SP cells. These studies suggest that nicotine might be promoting NSCLC growth and metastasis by inducing the secretion of SCF, and raise the possibility that targeting signalling cascades that activate E2F1 might be an effective way to combat NSCLC.

Endothelin-converting enzyme 1 and ß-arrestins exert spatiotemporal control of substance P-induced inflammatory signals.

Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins ß-arrestin (ßARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and ßARR1/2 terminate plasma membrane Ca(2+) signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. ßARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from ßARRs, to recycle. Thus, both ECE-1 and ßARRs mediate the resensitization of NK1R Ca(2+) signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or ßARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of ßARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation.

Nodal signals via ß-arrestins and RalGTPases to regulate trophoblast invasion.

Placentation is critical for establishing a healthy pregnancy. Trophoblasts mediate implantation and placentation and certain subtypes, most notably extravillous cytotrophoblast, are highly invasive. Trophoblast invasion is tightly regulated by microenvironmental cues that dictate placental morphology and depth. In choriocarcinomas, malignant trophoblast cells become hyperinvasive, breaching the myometrium and leading to major complications. Nodal, a member of the TGF-ß superfamily, is expressed throughout the endometrium during the peri-implantation period and in invasive trophoblast cells. Nodal promotes the invasion of numerous types of cancer cells. However, Nodal's role in trophoblast and choriocarcinoma cell invasion is unclear. Here we show that Nodal stimulates the invasion of both the non-malignant HTR-8SV/neo trophoblast and JAR choriocarcinoma cells in a dose-dependent manner. We found that endogenous ß-arrestins and Ral GTPases, key regulators of the cell cytoskeleton, are constitutively associated with Nodal receptors (ALK4 and ALK7) in trophoblast cells and that RalA is colocalized with ALK4 in endocytic vesicles. Nodal stimulates endogenous ß-arrestin2 to associate with phospho-ERK1/2, and knockdown of ß-arrestin or Ral proteins impairs Nodal-induced trophoblast and choriocarcinoma cell invasion. These results demonstrate, for the first time, that ß-arrestins and RalGTPases are important regulators of Nodal-induced invasion.

Alpha1a-adrenoceptor genetic variant induces cardiomyoblast-to-fibroblast-like cell transition via distinct signaling pathways.

The role of naturally occurring human α1a-Adrenergic Receptor (α1aAR) genetic variants associated with cardiovascular disorders is poorly understood. Here, we present the novel findings that expression of human α1aAR-247R (247R) genetic variant in cardiomyoblasts leads to transition of cardiomyoblasts into a fibroblast-like phenotype, evidenced by morphology and distinct de novo expression of characteristic genes. These fibroblast-like cells exhibit constitutive, high proliferative capacity and agonist-induced hypertrophy compared with cells prior to transition. We demonstrate that constitutive, synergistic activation of EGFR, Src and ERK kinases is the potential molecular mechanism of this transition. We also demonstrate that 247R triggers two distinct EGFR transactivation-dependent signaling pathways: 1) constitutive Gq-independent ß-arrestin-1/Src/MMP/EGFR/ERK-dependent hyperproliferation and 2) agonist-induced Gq- and EGFR/STAT-dependent hypertrophy. Interestingly, in cardiomyoblasts agonist-independent hyperproliferation is MMP-dependent, but in fibroblast-like cells it is MMP-independent, suggesting that expression of α1aAR genetic variant in cardiomyocytes may trigger extracellular matrix remodeling. Thus, these novel findings demonstrate that EGFR transactivation by α1aAR-247R leads to hyperproliferation, hypertrophy and alterations in cardiomyoblasts, suggesting that these unique genetically-mediated alterations in signaling pathways and cellular function may lead to myocardial fibrosis. Such extracellular matrix remodeling may contribute to the genesis of arrhythmias in certain types of heart failure.

Discovery and characterization of a G protein-biased agonist that inhibits ß-arrestin recruitment to the D2 dopamine receptor.

A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and ß-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate ß-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit ß-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated ß-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate ß-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate ß-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.

Sphingosine-1-phosphate-induced Flk-1 transactivation stimulates mouse embryonic stem cell proliferation through S1P1/S1P3-dependent ß-arrestin/c-Src pathways.

Although recent findings showed that the bioactive lipid metabolites can regulate the ES cell functions, the physiological relevance of interaction between sphingosine-1-phosphate (S1P) and Flk-1 and its related signaling molecules are not yet clear in ES cell proliferation. In the present study, S1P1-5 receptors were expressed in mouse ES cells and S1P increased S1P1-3 receptor expression level. S1P treatment stimulated the cellular proliferation in S1P1/3-dependent manner, located in lipid rafts. In response to S1P, ß-arrestin was recruited to S1P1/3 receptor and c-Src was activated. S1P also increased the binding of S1P1/3 receptor with Flk-1. Similar to responses for VEGF, S1P increased Flk-1 phosphorylation, which was blocked by ß-arrestin siRNA, and PP2, but not by VEGF-A164 antibody or VEGF siRNA. In addition, S1P induced VEGF expression and VEGFR2 kinase inhibitor (SU1498) blocked the S1P-induced cellular proliferation. However, VEGF-A164 antibody or VEGF siRNA partially blocked S1P-induced cellular proliferation, suggesting that both VEGF-dependent Flk-1 activation and VEGF-independent Flk-1 activation are involved in S1P-induced ES cell proliferation. S1P and VEGF-induced phosphorylation of ERK and JNK were blocked by pretreatment with SU1498. Moreover, inhibition of ERK and JNK blocked S1P-induced cellular proliferation. In conclusion, S1P-elicited transactivation of Flk-1 mediated by S1P1/3-dependent ß-arrestin/c-Src pathways stimulated mouse ES cell proliferation.

The bile acid receptor TGR5 does not interact with ß-arrestins or traffic to endosomes but transmits sustained signals from plasma membrane rafts.

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of ß-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with ß-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with ß-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-ß-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with ß-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.

The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting ß-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by ß-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on ß-arrestin 2 mediated internalization.

ß-Arrestins as potential therapeutic targets for Alzheimer's disease.

ß-arrestins represent a small family of G protein-coupled receptors (GPCRs) regulators, which provide modulating effects by facilitating desensitization and internalization of GPCRs as well as initiating their own signalings. Recent reports have demonstrated that ß-arrestins levels were correlated with amyloid-ß peptide (Aß) pathology in brains of Alzheimer's disease (AD) patients and animal models. ß-arrestins could enhance the activity of γ-secretase via interacting with anterior pharynx defective 1 subunit, which increased Aß production and contributed to the pathogenesis of AD. In addition, Aß-induced internalization of ß2-adrenergic receptor internalization and loss of dendritic spine in neurons were proven to be mediated by ß-arrestins, further establishing their pathogenic role in AD. More importantly, deletion of ß-arrestins markedly attenuated AD pathology, without causing any gross abnormality. Here, we review the evidence about the roles of ß-arrestins in the progression of AD. In addition, the established and postulated mechanisms by which ß-arrestins mediated in AD pathogenesis are also discussed. Based on the role of ß-arrestins in AD pathogenesis, genetically or pharmacologically targeting ß-arrestins might provide new opportunities for AD treatment.

Epigallocatechin gallate attenuated the activation of rat cardiac fibroblasts induced by angiotensin II via regulating ß-arrestin1.

BACKGROUND/AIMS: Angiotensin II (AngII) activated cardiac fibroblasts (CFs) predominantly through AngII subtype 1a receptor (AT1aR). This study was carried out to explore the potential inhibitory effects and mechanisms of epigallocatechin gallate (EGCG) on AngII induced rat CFs. METHODS: Viability, proliferation and collagen production of CFs were measured by MTT assay, [3H]-thymidine and [3H]-proline incorporation respectively. ß-arrestin1 (ßarr1), AT1aR and AT1bR mRNA levels were determined by quantitative PCR. AT1R, Gq, ßarr 1/2, phosphorylated kinase C (p-PKC)-delta expressions were detected by western blotting. We blocked ßarr1 expression using ßarr1 small interfering RNA (siRNA). RESULTS: EGCG inhibited the activation of CFs induced by AngII. ßarr1 mRNA level revealed a positive correlation with the viability of CFs. SiRNA targeting ßarr1 blocked the activation of CFs. In vitro, AngII increased ßarr1 mRNA, total and membrane ßarr1 protein expressions, but reduced AT1aR mRNA, global and membrane AT1R, total Gq and cytoplasmic p-PKC-delta levels. Administration of EGCG restored the above abnormalities, whereas Gq levels were not affected. CONCLUSION: Our findings showed that ßarr1 is essential for AngII-mediated activation of CFs. EGCG attenuated CFs activation induced by AngII via regulating ßarr1 and thus, modulating AT1aR mediated signaling.

Specific interactions between Epac1, ß-arrestin2 and PDE4D5 regulate ß-adrenergic receptor subtype differential effects on cardiac hypertrophic signaling.

ß1 and ß2 adrenergic receptors (ßARs) are highly homologous but fulfill distinct physiological and pathophysiological roles. Here we show that both ßAR subtypes activate the cAMP-binding protein Epac1, but they differentially affect its signaling. The distinct effects of ßARs on Epac1 downstream effectors, the small G proteins Rap1 and H-Ras, involve different modes of interaction of Epac1 with the scaffolding protein ß-arrestin2 and the cAMP-specific phosphodiesterase (PDE) variant PDE4D5. We found that ß-arrestin2 acts as a scaffold for Epac1 and is necessary for Epac1 coupling to H-Ras. Accordingly, knockdown of ß-arrestin2 prevented Epac1-induced histone deacetylase 4 (HDAC4) nuclear export and cardiac myocyte hypertrophy upon ß1AR activation. Moreover, Epac1 competed with PDE4D5 for interaction with ß-arrestin2 following ß2AR activation. Dissociation of the PDE4D5-ß-arrestin2 complex allowed the recruitment of Epac1 to ß2AR and induced a switch from ß2AR non-hypertrophic signaling to a ß1AR-like pro-hypertrophic signaling cascade. These findings have implications for understanding the molecular basis of cardiac myocyte remodeling and other cellular processes in which ßAR subtypes exert opposing effects.