Anti-Inflammatory and Pro-Resolving Actions of the N-Terminal Peptides Ac2-26, Ac2-12, and Ac9-25 of Annexin A1 on Conjunctival Goblet Cell Function.

Annexin A1 (AnxA1) is the primary mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca2+] ([Ca2+]i) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12, and Ac9-25. The increase in [Ca2+]i caused by AnxA1 and its N-terminal peptides in goblet cells was measured to determine the formyl peptide receptors used by the compounds and the action of the peptides on histamine stimulation. Changes in [Ca2+]i were determined by using a fluorescent Ca2+ indicator. AnxA1 and its peptides each activated formyl peptide receptors in goblet cells. AnxA1 and Ac2-26 at 10-12 mol/L and Ac2-12 at 10-9 mol/L inhibited the histamine-stimulated increase in [Ca2+]i, as did resolvin D1 and lipoxin A4 at 10-12 mol/L, whereas Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H1 receptor through the p42/p44 mitogen-activated protein kinase/extracellular regulated kinase 1/2, ß-adrenergic receptor kinase, and protein kinase C pathways, whereas Ac2-12 counter-regulated only through ß-adrenergic receptor kinase. In conclusion, current data show that the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca2+]i and counter-regulation of the H1 receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.

Specific and behaviorally consequential astrocyte Gq GPCR signaling attenuation in vivo with ißARK.

Astrocytes respond to neurotransmitters and neuromodulators using G-protein-coupled receptors (GPCRs) to mediate physiological responses. Despite their importance, there has been no method to genetically, specifically, and effectively attenuate astrocyte Gq GPCR pathways to explore consequences of this prevalent signaling mechanism in vivo. We report a 122-residue inhibitory peptide from ß-adrenergic receptor kinase 1 (ißARK; and inactive D110A control) to attenuate astrocyte Gq GPCR signaling. ißARK significantly attenuated Gq GPCR Ca2+ signaling in brain slices and, in vivo, altered behavioral responses, spared other GPCR responses, and did not alter astrocyte spontaneous Ca2+ signals, morphology, electrophysiological properties, or gene expression in the striatum. Furthermore, brain-wide attenuation of astrocyte Gq GPCR signaling with ißARK using PHP.eB adeno-associated viruses (AAVs), when combined with c-Fos mapping, suggested nuclei-specific contributions to behavioral adaptation and spatial memory. ißARK extends the toolkit needed to explore functions of astrocyte Gq GPCR signaling within neural circuits in vivo.

ß adrenergic receptor modulated signaling in glioma models: promoting ß adrenergic receptor-ß arrestin scaffold-mediated activation of extracellular-regulated kinase 1/2 may prove to be a panacea in the treatment of intracranial and spinal malignancy and extra-neuraxial carcinoma.

Neoplastically transformed astrocytes express functionally active cell surface ß adrenergic receptors (ßARs). Treatment of glioma models in vitro and in vivo with ß adrenergic agonists variably amplifies or attenuates cellular proliferation. In the majority of in vivo models, ß adrenergic agonists generally reduce cellular proliferation. However, treatment with ß adrenergic agonists consistently reduces tumor cell invasive potential, angiogenesis, and metastasis. ß adrenergic agonists induced decreases of invasive potential are chiefly mediated through reductions in the expression of matrix metalloproteinases types 2 and 9. Treatment with ß adrenergic agonists also clearly reduce tumoral neoangiogenesis, which may represent a putatively useful mechanism to adjuvantly amplify the effects of bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor receptor. We may accordingly designate ßagonists to represent an enhancer of bevacizumab. The antiangiogenic effects of ß adrenergic agonists may thus effectively render an otherwise borderline effective therapy to generate significant enhancement in clinical outcomes. ß adrenergic agonists upregulate expression of the major histocompatibility class II DR alpha gene, effectively potentiating the immunogenicity of tumor cells to tumor surveillance mechanisms. Authors have also demonstrated crossmodal modulation of signaling events downstream from the ß adrenergic cell surface receptor and microtubular polymerization and depolymerization. Complex effects and desensitization mechanisms of the ß adrenergic signaling may putatively represent promising therapeutic targets. Constant stimulation of the ß adrenergic receptor induces its phosphorylation by ß adrenergic receptor kinase (ßARK), rendering it a suitable substrate for alternate binding by ß arrestins 1 or 2. The binding of a ß arrestin to ßARK phosphorylated ßAR promotes receptor mediated internalization and downregulation of cell surface receptor and contemporaneously generates a cell surface scaffold at the ßAR. The scaffold mediated activation of extracellular regulated kinase 1/2, compared with protein kinase A mediated activation, preferentially favors cytosolic retention of ERK1/2 and blunting of nuclear translocation and ensuant pro-transcriptional activity. Thus, ßAR desensitization and consequent scaffold assembly effectively retains the cytosolic homeostatic functions of ERK1/2 while inhibiting its pro-proliferative effects. We suggest these mechanisms specifically will prove quite promising in developing primary and adjuvant therapies mitigating glioma growth, angiogenesis, invasive potential, and angiogenesis. We suggest generating compounds and targeted mutations of the ß adrenergic receptor favoring ß arrestin binding and scaffold facilitated activation of ERK1/2 may hold potential promise and therapeutic benefit in adjuvantly treating most or all cancers. We hope our discussion will generate fruitful research endeavors seeking to exploit these mechanisms.

ß Adrenergic Receptor Kinase C-Terminal Peptide Gene-Therapy Improves ß2-Adrenergic Receptor-Dependent Neoangiogenesis after Hindlimb Ischemia.

After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of ß2-adrenergic receptor (ß2AR) signaling, leading to reduced revascularization. Indeed, in vivo ß2AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of ßAR signaling, and ß adrenergic receptor kinase C-terminal peptide (ßARKct), a peptide inhibitor of GRK2, has been shown to prevent ßAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of ß2AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated ßARKct gene transfer in an experimental model of HI and its effects on ßAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant ßAR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the ßARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and ßAR density in the ischemic muscle, compared with control groups. The effect of ßARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the ßARKct fenoterol, a ß2AR-agonist, induced enhanced ß2AR proangiogenic signaling and increased EC function. Our results suggest that ßARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced ß2AR down-regulation.

Optodynamic simulation of ß-adrenergic receptor signalling.

Optogenetics has provided a revolutionary approach to dissecting biological phenomena. However, the generation and use of optically active GPCRs in these contexts is limited and it is unclear how well an opsin-chimera GPCR might mimic endogenous receptor activity. Here we show that a chimeric rhodopsin/ß2 adrenergic receptor (opto-ß2AR) is similar in dynamics to endogenous ß2AR in terms of: cAMP generation, MAP kinase activation and receptor internalization. In addition, we develop and characterize a novel toolset of optically active, functionally selective GPCRs that can bias intracellular signalling cascades towards either G-protein or arrestin-mediated cAMP and MAP kinase pathways. Finally, we show how photoactivation of opto-ß2AR in vivo modulates neuronal activity and induces anxiety-like behavioural states in both fiber-tethered and wireless, freely moving animals when expressed in brain regions known to contain ß2ARs. These new GPCR approaches enhance the utility of optogenetics and allow for discrete spatiotemporal control of GPCR signalling in vitro and in vivo.

Anchored protein kinase A signalling in cardiac cellular electrophysiology.

The cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is an elementary molecule involved in both acute and chronic modulation of cardiac function. Substantial research in recent years has highlighted the importance of A-kinase anchoring proteins (AKAP) therein as they act as the backbones of major macromolecular signalling complexes of the ß-adrenergic/cAMP/PKA pathway. This review discusses the role of AKAP-associated protein complexes in acute and chronic cardiac modulation by dissecting their role in altering the activity of different ion channels, which underlie cardiac action potential (AP) generation. In addition, we review the involvement of different AKAP complexes in mechanisms of cardiac remodelling and arrhythmias.

AAV6-ßARKct gene delivery mediated by molecular cardiac surgery with recirculating delivery (MCARD) in sheep results in robust gene expression and increased adrenergic reserve.

OBJECTIVE: Genetic modulation of heart function is a novel therapeutic strategy. We investigated the effect of molecular cardiac surgery with recirculating delivery (MCARD)-mediated carboxyl-terminus of the ß-adrenergic receptor kinase (ßARKct) gene transfer on cardiac mechanoenergetics and ß-adrenoreceptor (ßAR) signaling. METHODS: After baseline measurements, sheep underwent MCARD-mediated delivery of 10(14) genome copies of self-complimentary adeno-associated virus (scAAV6)-ßARKct. Four and 8 weeks after MCARD, mechanoenergetic studies using magnetic resonance imaging were performed. Tissues were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ßAR density, cyclic adenosine monophosphate levels, and physiologic parameters were evaluated. RESULTS: There was a significant increase in dP/dt(max) at 4 weeks: 1384 ± 76 versus 1772 ± 182 mm Hg/s; and the increase persisted at 8 weeks in response to isoproterenol (P < .05). Similarly, the magnitude of dP/dt(min) increased at both 4 weeks and 8 weeks with isoproterenol stimulation (P < .05). At 8 weeks, potential energy was conserved, whereas in controls there was a decrease in potential energy (P < .05) in response to isoproterenol. RT-qPCR confirmed robustness of ßARKct expression throughout the left ventricle and undetectable expression in extracardiac tissues. Quantitative Western blot data confirmed higher expression of ßARKct in the left ventricle: 0.46 ± 0.05 versus 0.00 in lung and liver (P < .05). Survival was 100% and laboratory parameters of major organ function were within normal limits. CONCLUSIONS: MCARD-mediated ßARKct delivery is safe, results in robust cardiac-specific gene expression, enhances cardiac contractility and lusitropy, increases adrenergic reserve, and improves energy utilization efficiency in a preclinical large animal model.

Gßγ subunits inhibit Epac-induced melanoma cell migration.

BACKGROUND: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein ßγ subunits (Gßγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gßγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gßγ and Epac in melanoma, which plays a role in regulation of cell migration. METHODS: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers. RESULTS: The effect of Gßγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gßγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-ß-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gßγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of ß1 and γ2, which is the major combination of Gßγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of ß adrenergic receptor kinase (ßARK-CT), an endogenous inhibitor for Gßγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gßγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of ß1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gßγ with ßARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPßS), a GDP analogue that inactivates Gßγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gßγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gßγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration. CONCLUSION: We found the cross talk of Ca 2+ signaling between Gßγ and Epac, which plays a major role in melanoma cell migration.

Negative regulation of Ca(2+) influx during P2Y(2) purinergic receptor activation is mediated by Gbetagamma-subunits.

We have previously reported that P2Y(2) purinoceptors and muscarinic M(3) receptors trigger Ca(2+) responses in HT-29 cells that differ in their timecourse, the Ca(2+) response to P2Y(2) receptor activation being marked by a more rapid decline of intracellular Ca(2+) concentration ([Ca(2+)](i)) after the peak response and that this rapid decline of [Ca(2+)](i) was slowed in cells expressing heterologous beta-adrenergic receptor kinase (betaARK). In the present study, we demonstrate that, during P2Y(2) receptor activation, betaARK expression increases the rate of Gd(3+)-sensitive Mn(2+) influx, a measure of the rate of store-operated Ca(2+) entry from the extracellular space, during P2Y(2) activation and that this effect of betaARK is mimicked by exogenous alpha-subunits of G(q), G(11) and G(i2). The effect of betaARK on the rate of Mn(2+) influx is thus attributable to its ability to scavenge G protein betagamma-subunits released during activation of P2Y(2) receptor. We further find that the effect of betaARK on the rate of Mn(2+) influx during P2Y(2) receptor activation can be overcome by arachidonic acid. In addition, the UTP-induced Mn(2+) influx rate was significantly increased by inhibitors of phospholipase A(2) (PLA(2)) and an siRNA directed against PLA(2)beta, but not by an siRNA directed against PLA(2)alpha or by inhibitors of arachidonic acid metabolism. These findings provide evidence for the existence of a P2Y(2) receptor-activated signalling system that acts in parallel with depletion of intracellular Ca(2+) stores to inhibit Ca(2+) influx across the cell membrane. This signalling process is mediated via Gbetagamma and involves PLA(2)beta and arachidonic acid.

Pasireotide and octreotide stimulate distinct patterns of sst2A somatostatin receptor phosphorylation.

Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.

Roles of c-Src in alpha1B-adrenoceptor phosphorylation and desensitization.

1 The role of the protein tyrosine kinase, c-Src, on the function and phosphorylation of alpha1B-adrenoceptors (alpha1B-AR) and their association with G-protein-coupled receptor kinase (GRK) isozymes was studied. 2 Inhibitors of this kinase (PP2 and Src Inhibitor II) decreased ( approximately 50-75%) noradrenaline- (NA) and phorbol myristate acetate-mediated receptor phosphorylation. Expression of a dominant-negative mutant of c-Src similarly reduced receptor phosphorylation induced by the natural agonists, active phorbol esters and endothelin-1 (ET-1). 3 c-Src, GRK2, GRK3 and GRK5 coimmunoprecipitate with alpha1B-ARs in the basal state. In cells treated with NA or phorbol myristate acetate the amount of coimmunoprecipitated GRK2 and GRK3 increased ( approximately 2- to 3-fold), while treatment with ET-1 only augmented the amount of coimmunoprecipitated GRK2 ( approximately 2-fold). The Src inhibitor, PP2, markedly attenuated all these increases. 4 Cell pretreatment with PP2 amplified the increase in intracellular-free calcium observed with NA, in the basal state and after the stimulation (desensitization) induced by ET-1. 5 The data suggest a role of c-Src in alpha1B-AR desensitization/phosphorylation and in the interaction of these ARs with GRKs.

Identification of Nogo as a novel indicator of heart failure.

Numerous genetically engineered animal models of heart failure (HF) exhibit multiple characteristics of human HF, including aberrant beta-adrenergic signaling. Several of these HF models can be rescued by cardiac-targeted expression of the Gbetagamma inhibitory carboxy-terminus of the beta-adrenergic receptor kinase (betaARKct). We recently reported microarray analysis of gene expression in multiple animal models of HF and their betaARKct rescue, where we identified gene expression patterns distinct and predictive of HF and rescue. We have further investigated the muscle LIM protein knockout model of HF (MLP-/-), which closely parallels human dilated cardiomyopathy disease progression and aberrant beta-adrenergic signaling, and their betaARKct rescue. A group of known and novel genes was identified and validated by quantitative real-time PCR whose expression levels predicted phenotype in both the larger HF group and in the MLP-/- subset. One of these novel genes is herein identified as Nogo, a protein widely studied in the nervous system, where it plays a role in regeneration. Nogo expression is altered in HF and normalized with rescue, in an isoform-specific manner, using left ventricular tissue harvested from both animal and human subjects. To investigate cell type-specific expression of Nogo in the heart, immunofluorescence and confocal microscopy were utilized. Nogo expression appears to be most clearly associated with cardiac fibroblasts. To our knowledge, this is the first report to demonstrate the relationship between Nogo expression and HF, including cell-type specificity, in both mouse and human HF and phenotypic rescue.

Confirmation of an association between the TNF(-308) promoter polymorphism and stroke risk in children with sickle cell anemia.

BACKGROUND AND PURPOSE: The etiology of stroke in children with sickle cell anemia (SCA) is complex and poorly understood. Growing evidence suggests that genetic factors beyond the sickle cell mutation influence stroke risk in SCA. We previously reported risk associations with polymorphisms in several proinflammatory genes in SCA children with ischemic stroke. The aim of this replication study was to confirm our previous findings of associations between the TNF(-308) G/A, IL4R 503 S/P, and ADRB2 27 Q/E polymorphisms and large vessel stroke risk. METHODS: Using previously collected MRA data, we assessed an independent population of SCA children from the multicenter Stroke Prevention Trial in Sickle Cell Anemia (STOP) for the presence or absence of large vessel stenosis. Samples were genotyped for 104 polymorphisms among 65 candidate vascular disease genes. Genotypic associations with risk of large vessel stroke were screened using univariable analysis and compared with results from our original study. Joint analysis of the 2 study populations combined was performed using multivariable logistic regression. RESULTS: A total of 96 children (49 MRA-positive, 47 MRA-negative) were included in this study. Of the SNP associations previously identified in the original study, the TNF(-308) G/A association with large vessel stroke remained significant and the IL4R 503 S/P variant approached significance in the joint analysis of the combined study populations. Consistent with our original findings, the TNF(-308) GG genotype was associated with a >3-fold increased risk of large vessel disease (OR=3.27; 95% CI=1.6, 6.9; P=0.006). Unadjusted analyses also revealed a previously unidentified association between the LTC4S(-444) A/C variant and large vessel stroke risk. CONCLUSIONS: Similar findings in 2 independent study populations strongly suggest that the TNF(-308) G/A promoter polymorphism is a clinically important risk factor for large vessel stroke in children with SCA. The previously observed association with the IL4R 503 S/P variant and the novel association with the LTC4S(-444) A/C variant suggest that these loci may also contribute to large vessel stroke risk in children with SCA.

Substrate specificities of g protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function.

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac beta-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes. Concentration-effect relationships of GRK2, GRK3, and their respective competitive inhibitors, GRK2ct and GRK3ct, at endogenous endothelin, alpha(1)-adrenergic, and beta(1)-adrenergic receptor-generated responses in cardiac myocytes were achieved by adenovirus gene transduction. GRK3 and GRK3ct were highly potent and efficient at the endothelin receptors (IC(50) for GRK3, 5 +/- 0.7 pmol/mg of protein; EC(50) for GRK3ct, 2 +/- 0.2 pmol/mg of protein). The alpha(1)-adrenergic receptor was also a preferred substrate of GRK3 (IC(50),7 +/- 0.4 pmol/mg of protein). GRK2 lacked efficacy at both endothelin and alpha(1)-adrenergic receptors despite massive overexpression. On the contrary, both GRK2ct and GRK3ct enhanced beta(1)-adrenergic receptor-induced cAMP production with comparable potencies. However, the potency of GRK3ct at beta(1)-adrenergic receptors was at least 20-fold lower than that at endothelin receptors. In conclusion, this study demonstrates distinct substrate specificities of GRK2 and GRK3 at different GPCRs in fully differentiated adult cardiac myocytes. As inferred from the above findings, GRK2 may play its primary role in regulation of cardiac contractility and chronotropy by controlling beta(1)-adrenergic receptors, whereas GRK3 may play important roles in regulation of cardiac growth and hypertrophy by selectively controlling endothelin and alpha(1)-adrenergic receptors.

The pharmacogenetics of lithium response depends upon clinical co-morbidity.

BACKGROUND: Based on results from randomized, controlled clinical trials, lithium monotherapy or lithium with the addition of an antipsychotic remains a first-line treatment option for both acute and long-term mood stabilization in bipolar mania. However, response to lithium is poor in bipolar patients who exhibit clinical characteristics such as rapid cycling and mixed manic states, suggesting that they may have a biologically and genetically distinct form of bipolar disorder. A test that could predict response to lithium based upon genetic factors would have significant clinical value. METHODS: Eight clinical characteristics were assessed in 92 lithium responders and 92 nonresponders; all probands were from families recruited for linkage studies. Lithium response was rated retrospectively from a standardized interviews and medical records. Eight candidate genes were selected from those reported to be associated with susceptibility to illness, lithium response, or lithium mechanism of action. Sixty-seven single nucleotide polymorphisms (SNPs) were genotyped in these subjects and analyzed for association with the defined clinical characteristics. RESULTS: Using q-value analysis for multiplicity correction, we found significant interactions between lithium response and SNPs (rs1387923 and rs1565445) in the gene encoding neurotrophic tyrosine kinase receptor type 2 (NTRK2) and suicidal ideation, and between SNP rs2064721 in the gene encoding inositol polyphosphate-1-phosphatase (INPP1) and post-traumatic stress disorder. CONCLUSION: These data support the idea that response to lithium has a multi-genetic etiology dependent upon manifestations of other clinical co-diagnoses.

Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2.

beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.

The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila.

Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. We show that Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. We found that Gprk2 function is required for transducing the Smo signal and that when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary.

Desensitization of the dopamine D1 and D2 receptor hetero-oligomer mediated calcium signal by agonist occupancy of either receptor.

When dopamine D1 and D2 receptors were coactivated in D1-D2 receptor hetero-oligomeric complexes, a novel phospholipase C-mediated calcium signal was generated. In this report, desensitization of this Gq/11-mediated calcium signal was demonstrated by pretreatment with dopamine or with the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297) or the D2-selective agonist quinpirole. Desensitization of the calcium signal mediated by D1-D2 receptor hetero-oligomers was initiated by agonist occupancy of either receptor subtype even though the signal was generated only by occupancy of both receptors. The efficacy, potency, and rate of calcium signal desensitization by agonist occupancy of the D1 receptor (t1/2, approximately 1 min) was far greater than by the D2 receptor (t1/2, approximately 10 min). Desensitization of the calcium signal was not mediated by depletion of calcium stores or internalization of the hetero-oligomer and was not decreased by inhibiting second messenger-activated kinases. The involvement of G protein-coupled receptor kinases 2 or 3, but not 5 or 6, in the desensitization of the calcium signal was shown, occurring through a phosphorylation independent mechanism. Inhibition of Gi protein function associated with D2 receptors increased D1 receptor-mediated desensitization of the calcium signal, suggesting that cross-talk between the signals mediated by the activation of different G proteins controlled the efficacy of calcium signal desensitization. Together, these results demonstrate the desensitization of a signal mediated only by hetero-oligomerization of two G protein-coupled receptors that was initiated by agonist occupancy of either receptor within the hetero-oligomer, albeit with differences in desensitization profiles observed.

Impaired p38 MAPK/HSP27 signaling underlies aging-related failure in opioid-mediated cardioprotection.

Cardioprotection and preconditioning mediated via G-protein-coupled receptors may be lost or impaired with advancing age, limiting ischemic tolerance and the ability to pharmacologically protect older hearts from ischemic injury. Our preliminary findings indicated a loss of delta-opioid receptor-mediated protection in aged vs. young mouse hearts, which may involve alterations in protective kinase signaling. In the present study, we tested the hypothesis that aging-related loss of opioid-triggered cardioprotection involves failure to activate p38 MAPK and its distal signaling targets. Langendorff-perfused hearts from young (10-14 weeks) or aged (24-26 months) C57 mice underwent 25-min ischemia and 45-min reperfusion in the presence or absence of 1 micromol/l DPDPE (delta-opioid agonist) or 1 micromol/l anisomycin (activator of p38 MAPK), and functional recovery and protein activation/phosphorylation were assessed. Contractile recovery was similar in untreated young and aged hearts (50+/-2% and 53+/-5%, respectively), and was enhanced by DPDPE in young hearts only (67+/-3%). Immunoblot analysis revealed that DPDPE comparably activated or phosphorylated GRK2, Akt, ERK1/2 and p70S6 kinase in young and aged hearts, whereas aging abrogated the stimulatory effects of DPDPE on p38 MAPK and HSP27. Treatment with anisomycin elicited comparable activation of p38 MAPK and HSP27 in both young and aged hearts, coupled with a pronounced and equivalent cardioprotection in the two groups (73+/-3% and 77+/-2%, respectively), an effect abolished by the p38 MAPK inhibitor, SB203580. These data indicate that aging-related loss of delta-opioid-mediated cardioprotection involves failure to activate p38 MAPK and HSP27. Direct targeting of this pathway elicits comparable protection in both age groups.

The membrane-proximal portion of CD3 epsilon associates with the serine/threonine kinase GRK2.

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 epsilon. Mass spectrometry analysis of CD3 epsilon-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 epsilon within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the alpha/beta TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 epsilon may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.