ß-Arrestin-1 protects against endoplasmic reticulum stress/p53-upregulated modulator of apoptosis-mediated apoptosis via repressing p-p65/inducible nitric oxide synthase in portal hypertensive gastropathy.

Portal hypertensive gastropathy (PHG) is a serious cause of bleeding in patients, and is associated with portal hypertension. ß-Arrestins (ß-arrestin-1 and ß-arrestin-2) are well-established mediators of endocytosis of G-protein-coupled receptors (GPCRs), ubiquitination, and G-protein-independent signaling. The role of ß-arrestin-1 (ß-arr1) in mucosal apoptosis in PHG remains unclear. The aim of this study was to investigate the involvement of ß-arr1 in PHG via its regulation of endoplasmic reticulum (ER) stress/p53-upregulated modulator of apoptosis (PUMA) apoptotic signaling. Gastric mucosal injury and apoptosis were studied in PHG patients and in PHG mouse models. The induction of ß-arr1 and the ER stress/PUMA signaling pathway were investigated, and the mechanisms of ß-arr1-regulated gastric mucosal apoptosis were analyzed in vivo and in vitro experiments. ß-arr1 and ER stress/PUMA signaling elements were markedly induced in the gastric mucosa of PHG patients and mouse models. Blockage of ER stress demonstrably attenuated the mucosal apoptosis of PHG, while targeted deletion of ß-arr1 significantly aggravated the injury and ER stress/PUMA-mediated apoptosis. ß-arr1 limited the activation of p65 to repress TNF-α-induced inducible nitric oxide synthase (iNOS) expression and NO release, which could regulate ER stress/PUMA-mediated mucosal apoptosis in PHG. In vivo and in vitro experiments further demonstrated that ß-arr1 protected against mucosal apoptosis by repressing TNF-α-induced iNOS expression via inhibiting the activation of p65. These results indicated that ß-arr1 regulated ER stress/PUMA-induced mucosal epithelial apoptosis through suppression of the TNF-α/p65/iNOS signaling pathway activation and that ß-arr1 is a potential therapeutic target for PHG.

Co-expression of ß-arrestin1 and NF-кB is associated with cancer progression and poor prognosis in lung adenocarcinoma.

ß-arrestin1 and NF-κB have been demonstrated to be associated with tumorigenesis, tumor progression, and metastasis. Thus far, there is nevertheless little study about these two molecules in lung adenocarcinoma. The aim of this study was to investigate the correlation between ß-arrestin1 and NF-κB expression and the clinicopathological characteristics in lung adenocarcinoma. A total of 115 surgically resected lung adenocarcinoma patients were recruited for the study. Expression of ß-arrestin1 and p65 were detected by immunohistochemistry (IHC) in lung adenocarcinoma tissue samples. Nuclear expression of ß-arrestin1 and p65 were observed in 39.1 % (45/115) and 46.1 % (53/115) cases of lung adenocarcinoma, respectively. And high expression of ß-arrestin1 had negative prognostic impact for overall survival (OS) and disease-free survival (DFS) (p = 0.034 and p = 0.033). In addition, overexpression of p65 indicated a significantly poor OS and DFS than those of lower-expression (p = 0.038 and p = 0.041). Furthermore, co-expression of nuclear ß-arrestin1 and p65 correlated with poorer OS and DFS in lung adenocarcinoma patients. Multivariate analysis using the Cox regression model confirmed that co-expression of nuclear ß-arrestin1 and p65 was an independent prognostic factor for tumor progression (p = 0.008). In conclusion, these data indicated that co-expression of nuclear ß-arrestin1 and p65 was a novel predictor for worse prognosis in patients with lung adenocarcinoma.

Arrestin expression in E. coli and purification.

Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in E. coli and purification of the tag-free wild-type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that overall salt concentration is maintained at or above physiological levels.

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide: key roles of alanine-5 and threonine-6 in receptor activation.

BACKGROUND AND PURPOSE: The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. EXPERIMENTAL APPROACH: CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. KEY RESULTS: Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by ninefold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY(1(a)) receptor. CONCLUSIONS AND IMPLICATIONS: Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

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.

Cannabinoid agonists increase the interaction between ß-Arrestin 2 and ERK1/2 and upregulate ß-Arrestin 2 and 5-HT(2A) receptors.

We have recently reported that selective cannabinoid 2 (CB(2)) receptor agonists upregulate 5-HT(2A) receptors by enhancing ERK1/2 signaling in prefrontal cortex (PFCx). Increased activity of cortical 5-HT(2A) receptors has been associated with several neuropsychiatric disorders such as anxiety and schizophrenia. Here we examine the mechanisms involved in this enhanced ERK1/2 activation in rat PFCx and in a neuronal cell model. Sprague-Dawley rats treated with a non-selective cannabinoid agonist (CP55940, 50µg/kg, 7 days, i.p.) showed enhanced co-immunoprecipitation of ß-Arrestin 2 and ERK1/2, enhanced pERK protein levels, and enhanced expression of ß-Arrestin 2 mRNA and protein levels in PFCx. In a neuronal cell line, we found that selective CB(2) receptor agonists upregulate ß-Arrestin 2, an effect that was prevented by selective CB(2) receptor antagonist JTE-907 and CB(2) shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis, ERK1/2, and the AP-1 transcription factor also prevented the cannabinoid receptor-induced upregulation of ß-Arrestin 2. Our results suggest that sustained activation of CB(2) receptors would enhance ß-Arrestin 2 expression possibly contributing to its increased interaction with ERK1/2, thereby driving the upregulation of 5-HT(2A) receptors. The CB(2) receptor-mediated upregulation of ß-Arrestin 2 would be mediated, at least in part, by an ERK1/2-dependent activation of AP-1. These data could provide the rationale for some of the adverse effects associated with repeated cannabinoid exposure and shed light on some CB(2) receptor agonists that could represent an alternative therapeutic because of their minimal effect on serotonergic neurotransmission.

Inhibitor of G protein-coupled receptor kinase 2 normalizes vascular endothelial function in type 2 diabetic mice by improving ß-arrestin 2 translocation and ameliorating Akt/eNOS signal dysfunction.

In type 2 diabetes, although Akt/endothelial NO synthase (eNOS) activation is known to be negatively regulated by G protein-coupled receptor kinase 2 (GRK2), it is unclear whether the GRK2 inhibitor would have therapeutic effects. Here we examined the hypotensive effect of the GRK2 inhibitor and its efficacy agonist both vascular (aortic) endothelial dysfunction (focusing especially on the Akt/eNOS pathway) and glucose intolerance in two type 2 diabetic models (ob/ob mice and nicotinamide+streptozotocin-induced diabetic mice). Mice were treated with a single injection of the GRK2 inhibitor or vehicle, and the therapeutic effects were compared by examining vascular function and by Western blotting. The GRK2 inhibitor lowered blood pressure in both diabetic models but not in their age-matched controls. The GRK2 inhibitor significantly improved clonidine-induced relaxation only in diabetic (ob/ob and DM) mice, with accompanying attenuations of GRK2 activity and translocation to the plasma membrane. These protective effects of the GRK2 inhibitor may be attributable to the augmented Akt/eNOS pathway activation (as evidenced by increases in Akt phosphorylation at Ser(473) and at Thr(308), and eNOS phosphorylation at Ser(1177)) and to the prevention of the GRK2 translocation and promotion of ß-arrestin 2 translocation to the membrane under clonidine stimulation. Moreover, the GRK2 inhibitor significantly improved the glucose intolerance seen in the ob/ob mice. Our work provides the first evidence that in diabetes, the GRK2 inhibitor ameliorates vascular endothelial dysfunction via the Akt/eNOS pathway by inhibiting GRK2 activity and enhancing ß-arrestin 2 translocation under clonidine stimulation, thereby contributing to a blood pressure-lowering effect. We propose that the GRK2 inhibitor may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.

Defining MC1R regulation in human melanocytes by its agonist α-melanocortin and antagonists agouti signaling protein and ß-defensin 3.

The melanocortin 1 receptor (MC1R), a G(s) protein-coupled receptor, has an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human ß-defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-melanocyte-stimulating hormone (α-MSH))-induced increase in the activities of adenylate cyclase and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C, increased, whereas exposure to UV radiation reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, whereas continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6, as well as ß-arrestin 1. Therefore, in addition to the MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV.

Cooling-increased phospho-ß-arrestin-1 and ß-arrestin-1 expression levels in 3T3-L1 adipocytes.

Cooling induces several responses that are modulated by molecular inhibitors and activators and receptor signaling. Information regarding potential targets involved in cold response mechanisms is still insufficient. We examined levels of the receptor-signaling mediator ß-arrestin-1 and phospho-Ser-412 ß-arrestin-1 in 3T3-L1 adipocytes exposed to 4-37 °C or treated with some molecular agents at 37°C. We also cooled cells with or without modification and signal-modulating agents. These conditions did not decrease cell viability, and western blot analysis revealed that exposure to 4 °C for 1.5h and to 28 and 32 °C for 24 and 48 h increased phospho-ß-arrestin-1 and ß-arrestin-1 levels and that exposure to 4 and 18 °C for 3 and 4.5h increased ß-arrestin-1 level. Serum removal and rewarming abolished ß-arrestin-1 alterations induced by cooling. Mithramycin A (a transcription inhibitor) treatment for 4 and 24h increased the level of ß-arrestin-1 but not that of phospho-ß-arrestin-1. The level of phospho-ß-arrestin-1 was increased by okadaic acid (a phosphatase inhibitor), decreased by epinephrine and aluminum fluoride (receptor-signaling modulators), and unaffected by N-ethylmaleimide (an alkylating agent) at 37 °C. N-Ethylmaleimide and the receptor-signaling modulators did not alter ß-arrestin-1 expression at 37 °C but impaired the induction of phospho-ß-arrestin-1 at 28 and 32 °C without affecting the induction of ß-arrestin-1. We show that cold-induced ß-arrestin-1 alterations are partially mimicked by molecular agents and that the responsive machinery for ß-arrestin-1 requires serum factors and N-ethylmaleimide-sensitive sites and is linked to rewarming- and receptor signaling-responsive machinery. Our findings provide helpful information for clarifying the cold-responsive machinery for ß-arrestin-1 and elucidating low-temperature responses.

ß-arrestin-1 participates in thrombosis and regulates integrin aIIbß3 signalling without affecting P2Y receptors desensitisation and function.

ß-arrestin-1 (ß-arr1) and ß-arrestin-2 (ß-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of ß-arr1 or ß-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in ß-arr1-/- and ß-arr2-/- platelets. In addition, deficiency in ß-arr1 or ß-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between ß-arr1 and ß-arr2 may explain these unchanged platelet responses. Interestingly, ß-arr1-/- but not ß-arr2-/- mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in ß-arr1-/- and ß-arr2-/- mice, suggesting no defect in haemostasis. ß-arr1-/- platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbß3. ß-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbß3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for ß-arr1 in promoting thrombus formation, in part through its participation in αIIbß3 signalling, and no role of ß-arr1 and ß-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.

Increased expression of beta-arrestin 1 and 2 in murine models of rheumatoid arthritis: isoform specific regulation of inflammation.

Pro-inflammatory cytokines and chemokines play critical roles in autoimmune diseases including rheumatoid arthritis (RA). Recently, it has been reported that ß-arrestin 1 and 2 are involved in the regulation of inflammation. We hypothesized that ß-arrestin 1 and 2 play critical roles in murine models of RA. Using a collagen-induced arthritis (CIA) and a human TNFα transgenic (TNFtg) mouse model, we demonstrated that ß-arrestin 1 and 2 expression are significantly increased in joint tissue of CIA mice and TNFtg mice. In fibroblast-like synoviocytes (FLS) isolated from hind knee joint of CIA mice, we observed an increase of ß-arrestin 1 and 2 protein and mRNA levels in the early stage of arthritis. In FLS, low molecular weight hyaluronan (HA)-induced TNFα and IL-6 production was increased by overexpression of ß-arrestin 1 but decreased by overexpression of ß-arrestin 2 demonstrating isoform specific regulation. TNFα and HA induced an increase of ß-arrestin 1 and 2 expression in FLS, while high mobility group box (HMGB)-1 only stimulated ß-arrestin 1 expression. TNFα- or HA-induced ß-arrestin 2 expression was blocked by a p38 inhibitor. To examine the in vivo role of ß-arrestin 2 in the pathogenesis of arthritis, WT and ß-arrestin 2 KO mice were subjected to collagen antibody-induced arthritis (CAIA). ß-Arrestin 2 KO mice exhibited more severe arthritis in CAIA. Thus ß-arrestin 2 is anti-inflammatory in CAIA. These composite observations suggest that ß-arrestin 1 and 2 differentially regulate FLS inflammation and increased ß-arrestin 2 may reduce experimental arthritis severity.

Reduced expression of G protein-coupled receptor kinases in schizophrenia but not in schizoaffective disorder.

Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting a powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed a reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. A reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder.

ß-arrestin-2 regulation of the cAMP response element binding protein.

Previous work demonstrated that cystic fibrosis (CF) cells exhibit an increase in cAMP-mediated signaling as a characteristic response to lost CFTR function. Evidence for increased cAMP-mediated signaling in CF included increased phosphorylation of the cAMP response element binding protein (CREB) and elevated ß-arrestin-2 (ßarr2) expression. However, subsequent studies reveal that CREB activation in CF cells is independent of protein kinase-A (PKA). The goal of this study is to test the hypothesis that elevated ßarr2 expression leads to increased CREB activation in a PKA-independent mechanism. ßarr2-GFP expressing tracheal epithelial cells (ßarr2-GFP) exhibit an increase of pCREB content and subsequent CRE activation compared to GFP expressing control cells. ßarr2 activation of the ERK cascade represents a candidate mechanism leading to CREB activation. ERK exhibits increased activation in ßarr2-GFP cells compared to cont-GFP cells, and ERK inhibition diminishes CRE activation in both GFP and ßarr2-GFP cells. To test directly whether CREB regulation in CF is ßarr2-dependent, nasal epithelium excised from wt mice (Cftr +/+; ßarr2 +/+), CF mice (Cftr -/-; ßarr2 +/+), and DKO mice (Cftr -/-; ßarr2 -/-) were analyzed for pCREB protein content. Removal of ßarr2 expression from CF mice reduces both pCREB and pERK content to wt levels. These data indicate that CF-related CREB regulation is mediated directly through ßarr2 expression via the ERK pathway.

Arrestin-1 expression level in rods: balancing functional performance and photoreceptor health.

In rod photoreceptors, signaling persists as long as rhodopsin remains catalytically active. Phosphorylation by rhodopsin kinase followed by arrestin-1 binding completely deactivates rhodopsin. Timely termination prevents excessive signaling and ensures rapid recovery. Mouse rods express arrestin-1 and rhodopsin at ∼0.8:1 ratio, making arrestin-1 the second most abundant protein in the rod. The biological significance of wild type arrestin-1 expression level remains unclear. Here we investigated the effects of varying arrestin-1 expression on its intracellular distribution in dark-adapted photoreceptors, rod functional performance, recovery kinetics, and morphology. We found that rod outer segments isolated from dark-adapted animals expressing arrestin-1 at wild type or higher level contain much greater fraction of arrestin-1 than previously estimated, 15-25% of the total. The fraction of arrestin-1 residing in the outer segments (OS) in animals with low expression (4-12% of wild type) is much lower, 5-7% of the total. Only 4% of wild type arrestin-1 level in the outer segments was sufficient to maintain near-normal retinal morphology, whereas rapid recovery required at least ∼12%. Supra-physiological arrestin-1 expression improved light sensitivity and facilitated photoresponse recovery, but was detrimental for photoreceptor health, particularly in the peripheral retina. Thus, physiological level of arrestin-1 expression in rods reflects the balance between short-term functional performance of photoreceptors and their long-term health.

Beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent 5-HT4 receptor signalling.

G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein-independent 5-HT(4) receptor (5-HT(4)R)-operated Src/ERK (extracellular signal-regulated kinase) pathway, but not the G(s) pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor' C-terminus in both human embryonic kidney (HEK)-293 cells and colliculi neurons. This inhibition required two sequences of events: the association of beta-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-t domain and the phosphorylation, by GRK5, of beta-arrestin1 (at Ser(412)) bound to the receptor. Phosphorylated beta-arrestin1 in turn prevented activation of Src constitutively bound to 5-HT(4)Rs, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent signalling.

Beta-arrestin/Ral signaling regulates lysophosphatidic acid-mediated migration and invasion of human breast tumor cells.

The lipid mediator lysophosphatidic acid (LPA) plays a role in cancer progression and signals via specific G protein-coupled receptors, LPA(1-3). LPA has been shown to enhance the metastasis of breast carcinoma cells to bone. However, the mechanisms by which LPA receptors regulate breast cancer cell migration and invasion remain unclear. Breast cancer cell proliferation has been shown to be stimulated by Ral GTPases, a member of the Ras superfamily. Ral activity can be regulated by the multifunctional protein beta-arrestin. We now show that HS578T and MDA-MB-231 breast cancer cells and MDA-MB-435 melanoma cells have higher expression of beta-arrestin 1 mRNA compared with the nontumorigenic mammary MCF-10A cells. Moreover, we found that the mRNA levels of LPA1, LPA2, beta-arrestin 2, and Ral GTPases are elevated in the advanced stages of breast cancer. LPA stimulates the migration and invasion of MDA-MB-231 cells, but not of MCF-10A cells, and this is mediated by pertussis toxin-sensitive G proteins and LPA1. However, ectopic expression of LPA1 in MCF-10A cells caused these cells to acquire an invasive phenotype. Gene knockdown of either beta-arrestin or Ral proteins significantly impaired LPA-stimulated migration and invasion. Thus, our data show a novel role for beta-arrestin/Ral signaling in mediating LPA-induced breast cancer cell migration and invasion, two important processes in metastasis.

Morphine promotes apoptosis via TLR2, and this is negatively regulated by beta-arrestin 2.

We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that beta-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and beta-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of beta-arrestin 2 by transfecting beta-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and beta-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.

Endosomal colocalization of melanocortin-3 receptor and beta-arrestins in CAD cells with altered modification of AKT/PKB.

The melanocortin 3-receptor is involved in regulating energy metabolism, body fluid composition and inflammatory responses. Melanocortin receptors function by activating membrane bound adenylate cyclase. However, the literature reports indicate that some G protein coupled receptors (GPCRs) can also activate mitogen activated protein kinase (MAPK) or phosphoinositide 3 kinase (PI3K) signaling pathways consequent to their endocytosis. These studies were undertaken to evaluate the role of these pathways in MC3R signaling in brain-stem neuronal cells. Recruitment of arrestins is implicated in the activation of secondary pathways by GPCRs and our data shows the colocalization of either arrestin B1 or B2 with MC3R in endosomes. An alteration in PKB phosphorylation pattern was observed in MC3R expressing cells independent of agonist stimulation. MC3R transfectants exhibited increased proliferation rates and inhibition of PKB pathway with triciribine abrogated cell proliferation in both vector control and MC3R transfectants. PKB is constitutively active in proliferating CAD cells but could be further activated by culturing the cells in differentiation medium. These studies suggest that the AKT/PKB pathway plays an important role in the proliferation of CAD cells and suggest a link between MC3R and cell growth pathways that may involve the alteration of AKT/PKB signaling pathway.

Subcellular translocation of the eGFP-tagged TRPL channel in Drosophila photoreceptors requires activation of the phototransduction cascade.

Signal-mediated translocation of transient receptor potential (TRP) channels is a novel mechanism to fine tune a variety of signaling pathways including neuronal path finding and Drosophila photoreception. In Drosophila phototransduction the cation channels TRP and TRP-like (TRPL) are the targets of a prototypical G protein-coupled signaling pathway. We have recently found that the TRPL channel translocates between the rhabdomere and the cell body in a light-dependent manner. This translocation modifies the ion channel composition of the signaling membrane and induces long-term adaptation. However, the molecular mechanism underlying TRPL translocation remains unclear. Here we report that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, whereas TRPL-eGFP translocation could be directly visualized in intact eyes. TRPL-eGFP failed to translocate to the cell body in flies carrying severe mutations in essential phototransduction proteins, including rhodopsin, Galphaq, phospholipase Cbeta and the TRP ion channel, or in proteins required for TRP function. Our data, furthermore, show that the activation of a small fraction of rhodopsin and of residual amounts of the Gq protein is sufficient to trigger TRPL-eGFP internalization. In addition, we found that endocytosis of TRPL-eGFP occurs independently of dynamin, whereas a mutation of the unconventional myosin III, NINAC, hinders complete translocation of TRPL-eGFP to the cell body. Altogether, this study revealed that activation of the phototransduction cascade is mandatory for TRPL internalization, suggesting a critical role for the light induced conductance increase and the ensuing Ca2+ -influx in the translocation process. The critical role of Ca2+ influx was directly demonstrated when the light-induced TRPL-eGFP translocation was blocked by removing extracellular Ca2+.

Human melanocortin receptor 2 expression and functionality: effects of protein kinase A and protein kinase C on desensitization and internalization.

The aim of this study was to investigate the short-term regulation of the ACTH receptor human (h) melanocortin receptor 2 (MC2R) by transfection of a c-Myc-tagged hMC2R in the M3 cell line and assess its membrane expression by indirect immunofluorescence. Stimulation with ACTH induced production of cAMP with EC(50) values ranging from 7.6-11.9 nM in transient and stable transfectants, respectively. Pretreatment with ACTH induced a dose-dependent loss of cAMP production, from 1 pm up to 10 nM. Desensitization was also time dependent, with 70% loss of maximal responsiveness occurring after 15-min pretreatment with 10 nM ACTH, followed by a plateau up to 60 min. The decrease in hMC2R responsiveness was abrogated by individual treatment with protein kinase A (PKA) or protein kinase C inhibitors, H-89 and GF109203X. However, when added simultaneously, receptor responsiveness was raised over the maximal hMC2R activity observed in control cells. ACTH-induced loss of cAMP production was accompanied by receptor sequestration into intracellular vesicles (maximum after 30-min exposure). Cotransfection of M3 cells with the c-Myc-tagged hMC2R and beta-arrestin-2-green fluorescence protein along with sucrose treatment revealed that beta-arrestin-2-green fluorescence protein and c-Myc-hMC2R were redistributed in similar intracellular vesicles through a clathrin-dependent, but caveolae-independent, process. Sucrose pretreatment blocked receptor desensitization, indicating that hMC2R desensitization and internalization are interrelated. Moreover, preincubation with H-89 abrogated hMC2R internalization, whereas GF109203X had no effect. In conclusion, the present results indicate that PKA and protein kinase C act synergistically to induce hMC2R desensitization, but only PKA is essential for receptor internalization, highlighting the complex nature of the short-term regulatory pattern of this receptor.