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.

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.

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.

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.

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.

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.

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.

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.

beta-Arrestin 2 expression determines the transcriptional response to lysophosphatidic acid stimulation in murine embryo fibroblasts.

G protein-coupled receptors often employ novel signaling mechanisms, such as transactivation of epidermal growth factor (EGF) receptors or G protein-independent signals transmitted by beta-arrestins, to control the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In this study we investigated the role of beta-arrestins in lysophosphatidic acid (LPA) receptor-stimulated ERK1/2 activation using fibroblast lines derived from wild type, beta-arrestin 1, beta-arrestin 2, and beta-arrestin 1/2 knock-out mice. LPA stimulation produced robust ERK1/2 phosphorylation in all four backgrounds. In cells lacking beta-arrestin 2, >80% of LPA-stimulated ERK1/2 phosphorylation was mediated by transactivated EGF receptors. In contrast, ERK1/2 activation in cells expressing beta-arrestin 2 was predominantly EGF receptor-independent. Introducing FLAG epitope-tagged beta-arrestin 2 into the beta-arrestin 1/2 null background restored EGF receptor-independent ERK1/2 activation, indicating that beta-arrestin 2 expression confers ERK1/2 activation via a distinct mechanism. To determine the contributions of beta-arrestin 2, transactivated EGF receptors, and ERK1/2 to LPA-stimulated transcriptional responses, we employed gene expression arrays containing cDNA markers for G protein-coupled receptor-mediated signaling. In the beta-arrestin 1/2 null background, 1 h of exposure to LPA significantly increased transcription of seven marker genes. Six of these responses were EGF receptor-dependent, and two required ERK1/2 activation. In beta-arrestin 2 expressing cells, three of the seven LPA-stimulated transcriptional responses observed in the beta-arrestin 1/2 null background were lost. The four residual responses were independent of EGF receptor transactivation, but all were ERK1/2-dependent. These data indicate that beta-arrestin 2 functions both to attenuate EGF receptor transactivation-dependent signaling and to promote a distinct subset of ERK1/2-mediated responses to LPA receptor activation.

Regulated expression of G protein-coupled receptor kinases (GRK's) and beta-arrestins in osteoblasts.

Desensitization of G-protein coupled receptors (GPCR's) is largely mediated by a family of enzymes and protein co-factors termed GRKs and arrestins, respectively. In the present studies, we investigated expression of GRKs and arrestins in osteoblastic cell lines concentrating on the enzymes (GRK2 and GRK3) and protein co-factors (beta-arrestint 1 and beta-arrestin 2) that play dominant roles in regulating GPCR responsiveness in most tissues and cell types. We found that osteoblastic cells express similar amounts of GRK2 with either undetectable or lesser amounts of GRK3. In contrast, expression of beta-arrestin 1 and beta-arrestin 2 by osteoblastic cells varied between cell lines. To determine if GRK2 or beta-arrestin expression is modulated during osteoblast development, we assessed expression of GRK2 and beta-arrestin proteins during differentiation of the mouse osteoblastic cell line MC3T3-E1 cells over a 21-day period. We found that expression of GRK2 and beta-arrestin 2 increased to maximal levels by day 7 and then decreased 4-fold by day 21. In contrast, expression of beta-arrestin 1 increased to maximal levels by day 14 and then decreased 2-fold by day 21. Over this same time period (days 7-21), PTH/PTHrP receptor number decreased to a greater extent than the decrease in PTH(1-34)-induced cAMP generation, suggesting that responsiveness of individual PTH/PTHrP receptors was enhanced in differentiated cells. We conclude that (1) osteoblastic cell lines differentially express the enzymes and protein co-factors that modulate GPCR responsiveness and (2) expression of both GRK2 and beta-arrestins is temporally regulated during osteoblast development. These data are consistent with the notion that GPCR responsiveness may be differentially regulated in osteoblastic cell lines and during osteoblast development.

Changes in the G-protein-coupled receptor desensitization machinery during relapsing-progressive experimental allergic encephalomyelitis.

G-protein-coupled receptors (GPCR) play an important role in inflammation. Their responsiveness is regulated by G-protein-coupled receptor kinases (GRKs) and beta-arrestins. We show here that induction of experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG) resulted in a profound decrease in GRK2 and GRK6 protein in splenocytes during all phases of disease. GRK2 mRNA was also lower during EAE, although the decrease in mRNA was less pronounced than the decrease in GRK2 protein. Interestingly, beta-arrestin protein expression was significantly increased. Downregulation of GRK2 was restricted to the spleen and mesenteric lymph nodes and was not observed in peritoneal macrophages. Furthermore, EAE did not induce alterations in GRK2 expression in heart, liver and pituitary.

Interferon beta-1a counteracts effects of activation on the expression of G-protein-coupled receptor kinases 2 and 3, beta-arrestin-1, and regulators of G-protein signalling 2 and 16 in human mononuclear leukocytes.

Activation regulates the responsiveness of G-protein-coupled receptors (GPCRs) on T cells, and modifications in the activity of GPCRs characterize lymphocytes from some immune disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Some lines of evidence suggest that such an effect is connected with the altered expression of some GPCRs regulatory proteins. Herein we demonstrate that phitoemagglutinin (PHA)-induced activation leads to differential expression of G-protein-coupled receptor kinase (GRK) 2, GRK3, beta-arrestin-1, regulators of G-protein signalling (RGS) 2, and RGS16 and decreases responsiveness of mononuclear leukocytes (MNL) to the beta-adrenergic agonist isoproterenol. Interferon beta-1a (IFN beta-1a), which is known to ameliorate the course of MS, counteracts the activation-induced effects on the expression of these GPCR regulatory proteins in MNL. Furthermore, IFN beta-1a quenches the effects of PHA on the isoproterenol-induced accumulation of cyclic AMP (cAMP). We suggest that regulation of GPCRs responsiveness may be a relevant property of IFN beta-1a in MS.

Characterization of lymphocyte beta 2-adrenoceptor signalling in patients with left ventricular volume overload disease.

1. Studies using animal experimental models have suggested that the beta2-adrenoceptor is uncoupled in association with alterations in the expression of G-protein-coupled receptor kinases (GRK) 2/3 in heart failure. However, the functional expression of the components of this pathway in human disease has not been fully elucidated yet. In the present study, we evaluated the possibility that the regulation of beta2-adrenoceptor signalling components in patients with left ventricular volume overload (VOL) depends on the severity of the overload. 2. We characterized the lymphocyte GRK 2-6, beta-arrestins 1 and 2, beta2-adrenoceptor expression at the mRNA and protein levels, as well as the activity of adenylyl cyclase, protein kinases (PK) A and PKC in patients with VOL using healthy blood donors as controls. 3. In the patient group, GRK2 mRNA was increased by 61% (P < 0.001), GRK3 was increased by 54% (P < 0.005), GRK5 was increased fivefold (P < 0.001) and the beta-arrestin 2 mRNA was increased by 40% (P < 0.05). These increases were paralleled with a sixfold increase in GRK2, a twofold increase in GRK3 and a 1.3-fold increase in GRK5 protein levels. These changes were associated with a significant decrease in beta2-adrenoceptor mRNA, the basal, catalytic and receptor-mediated activity of adenylyl cyclase and sensitization of the forskolin-stimulated activity towards augmented inhibition by guanylimidodiphosphate. In general, the increase in GRK2 and 5 mRNA exhibited a positive correlation with the gravity of the haemodynamic load, as determined by changes in left ventricular fractional shortening. 4. The results suggest that VOL induces an increase in the expression of lymphocyte beta2-adrenoceptor-specific GRK and beta-arrestin 2 in association with an attenuation in beta2-adrenoceptor levels. It can be speculated that the cardiac circulatory system adapts itself to altered haemodynamic functional demands partly by altering beta2-adrenoceptor signalling.

Changes in the expression of G protein-coupled receptor kinases and beta-arrestin 2 in rat brain during opioid tolerance and supersensitivity.

We previously demonstrated that chronic treatment of rats with the mu-opioid receptor agonist sufentanil induced pharmacological tolerance associated with mu-opioid receptor desensitization and down-regulation. Administration of the calcium channel blocker nimodipine during chronic treatment with sufentanil prevented mu-opioid receptor down-regulation, induced down-stream supersensitization, and produced supersensitivity to the opioid effects. The focus of the present study was to determine a role for G protein-coupled receptor kinases (GRKs) and beta-arrestin 2 in agonist-induced mu-opioid receptor signalling modulation during chronic opioid tolerance and supersensitivity. Tolerance was induced by 7-day chronic infusion of sufentanil (2 microgram/h). Supersensitivity was induced by concurrent infusion of sufentanil (2 microgram/h) and nimodipine (1 microgram/h) for 7 days. Antinociception was evaluated by the tail-flick test. GRK2, GRK3, GRK6 and beta-arrestin 2 immunoreactivity levels were determined by western blot in brain cortices. Acute and chronic treatment with sufentanil induced analgesic tolerance, associated with up-regulation of GRK2, GRK6, and beta-arrestin 2. GRK3 expression only was increased in the acutely treated group. When nimodipine was associated to the chronic opioid treatment, tolerance expression was prevented, and immunoreactivity levels of GRK2, GRK6 and beta-arrestin 2 recovered the control values. These data indicate that GRK2, GRK3, GRK6 and beta-arrestin 2 are involved in the short- and long-term adaptive changes in mu-opioid receptor activity, contributing to tolerance development in living animals. These observations also suggest that GRKs and beta-arrestin 2 could constitute pharmacological targets to prevent opioid tolerance development, and to improve the analgesic efficacy of opioid drugs.

Adjuvant arthritis induces down-regulation of G protein-coupled receptor kinases in the immune system.

G protein-coupled receptors (GPCR) play a crucial role in the regulation of the immune response by, e.g., chemokines, PGs, and beta(2)-adrenergic agonists. The responsiveness of these GPCRs is turned off by the family of G protein-coupled receptor kinases (GRK1-6). These kinases act by phosphorylating the GPCR in an agonist-dependent manner, resulting in homologous desensitization of the receptor. Although GRKs are widely expressed throughout the body, leukocytes express relatively high levels of GRKs, in particular GRK2, -3, and -6. We investigated whether in vivo the inflammatory disease adjuvant arthritis (AA) induces changes in GRK expression and function in the immune system. In addition, we analyzed whether the systemic effects of AA also involve changes in GRKs in nonimmune organs. At the peak of the inflammatory process, we observed a profound down-regulation of GRK2, -3, and -6 in splenocytes and mesenteric lymph node cells from AA rats. Interestingly, no changes in GRK were observed in thymocytes and in nonimmune organs such as heart and pituitary. During the remission phase of AA, GRK levels in spleen and mesenteric lymph nodes are returning to baseline levels. The decrease in GRK2 at the peak of AA is restricted to CD45RA(+) B cells and CD4(+) T cells, and was not observed in CD8(+) T cells. In conclusion, we demonstrate in this study, for the first time, that an inflammatory process in vivo induces a tissue-specific down-regulation of GRKs in the immune system.

Expression of beta-arrestins in toxic and cold thyroid nodules.

beta-Arrestins mediate agonist dependent desensitization of G protein-coupled receptors. Somatic TSH receptor mutations were identified in the majority of hot thyroid nodules. When transiently overexpressed in COS 7 cells these mutations resulted in constitutive activation of the cAMP pathway. However, the in vivo mechanisms and the in vivo desensitization of these TSH receptor mutations are unknown. Moreover, constitutively activated beta-adrenergic receptors are known to be constitutively desensitized. Therefore, we investigated the expression of beta-arrestins in toxic thyroid nodules (TTNs) with and without somatic TSH receptor mutation and in cold thyroid nodules (CTNs) by Western blotting and ELISA. Expression of beta-arrestin 2 was increased in all TTNs while beta-arrestin 2 expression was decreased in CTNs compared to their corresponding surrounding tissue. The mean beta-arrestin 1 expression was unchanged in the cytosol of TTNs, in membranes and cytosol of CTNs and decreased in the membranes of TTNs compared to their surrounding tissue. Transient coexpression of beta-arrestins 1 or 2 with the TSH receptor in HEK 293 cells and subsequent determination of cAMP showed that in vitro both beta-arrestins interact with the TSH receptor and are able to desensitize the receptor. The increased beta-arrestin 2 expression in TTNs and the desensitization of the TSH receptor by beta-arrestin 2 in vitro suggest that the beta-arrestin 2 expression is cAMP dependent and that beta-arrestin 2 very likely desensitizes the constitutively activated TSH receptor in toxic thyroid nodules.

Expression patterns of the regulatory proteins G protein-coupled receptor kinase 2 and beta-arrestin 1 during rat postnatal brain development: effect of hypothyroidism.

G protein-coupled receptor kinase 2 (GRK2) and beta-arrestin 1 are key regulatory proteins that modulate the desensitization and resensitization of a wide variety of G protein-coupled receptors (GPCRs) involved in brain functions. In this report, we describe the postnatal developmental profile of the mRNA and protein levels of GRK2 and beta-arrestin 1 in rat brain. The expression levels of GRK2 and beta-arrestin 1 display a marked increase at the second and third week after birth, respectively, consistent with an involvement of these proteins in brain maturation processes. However, the expression attained at birth and during the first postnatal week with respect to adult values (45-70% for GRK2, approximately 30% for beta-arrestin 1) is relatively high compared to that reported for several GPCRs, indicating the existence of changes in the ratio of receptors to their regulatory proteins during brain development. On the other hand, we report that experimental hypothyroidism results in changes in the patterns of expression of GRK2 and beta-arrestin 1 in cerebral cortex, leading to a 25-30% reduction in GRK2 levels at several stages of development. Such changes could help to explain the alterations in GPCR signaling that occur during this pathophysiological condition.

Role of G protein-coupled receptor kinases in glucose-dependent insulinotropic polypeptide receptor signaling.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response was abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. Beta-arrestin-1 transfection also induced a significantly decrease in GIP-stimulated cAMP production, and this effect was greater with cotransfection of both GRK2 and beta-arrestin-1 than with either alone. In betaTC3 cells, expression of GRK2 or beta-arrestin-1 attenuated GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion was not affected. GRK2 and beta-arrestin-1 messenger RNAs were identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells. Overexpression of GRK2 enhanced agonist-induced GIPR phosphorylation, but receptor endocytosis was not affected by cotransfection with GRKs or beta-arrestin-1. These results suggest a potential role for GRK2/beta-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR.