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After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of β2-adrenergic receptor (β2AR) signaling, leading to reduced revascularization. Indeed, in vivo β2AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of βAR signaling, and β adrenergic receptor kinase C-terminal peptide (βARKct), a peptide inhibitor of GRK2, has been shown to prevent βAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of β2AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated βARKct gene transfer in an experimental model of HI and its effects on βAR 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 βAR 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 βARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and βAR density in the ischemic muscle, compared with control groups. The effect of βARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the βARKct fenoterol, a β2AR-agonist, induced enhanced β2AR proangiogenic signaling and increased EC function. Our results suggest that βARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced β2AR down-regulation., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

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 β-adrenergic receptor kinase (βARKct) gene transfer on cardiac mechanoenergetics and β-adrenoreceptor (βAR) signaling., Methods: After baseline measurements, sheep underwent MCARD-mediated delivery of 10(14) genome copies of self-complimentary adeno-associated virus (scAAV6)-βARKct. 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. βAR 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 βARKct expression throughout the left ventricle and undetectable expression in extracardiac tissues. Quantitative Western blot data confirmed higher expression of βARKct 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 βARKct 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., (Copyright © 2012 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

This study was undertaken to determine alterations in the β-adrenoceptor (β-AR) signaling system in male and female rats at 4 weeks after the induction of arteriovenous (AV) fistula or shunt. AV shunt produced a greater degree of cardiac hypertrophy and larger increase in cardiac output in male than in female animals. Increases in plasma levels of norepinephrine and epinephrine (EPI) due to AV shunt were also higher in male than females. While no difference in the β(1)-AR affinity was seen in males and females, AV shunt induced increase in β(1)-AR density in female rats was higher than that in males. Furthermore, no changes in basal adenylyl cyclase (AC) V/VI mRNA levels were seen; however, the increase in EPI-stimulated AC activities was greater in AV shunt females than in males. AV shunt decreased myocardial β(1)-AR mRNA level in male rats and increased β(2)-AR mRNA level in female hearts; an increase in G(i)-protein mRNA was detected only in male hearts. Although GRK2 gene expression was increased in both sexes, an increase in GRK3 mRNA was seen only in AV shunt female rats. β-arrestin1 mRNA was elevated in females whereas β-arrestin 2 gene expression was increased in both male and female AV shunt rats. While these data demonstrate gender associated differences in various components of the β-AR system in cardiac hypertrophy due to AV shunt, only higher levels of plasma catecholamines may account for the greater increase in cardiac output and higher degree of cardiac hypertrophy in males.

Evidence from Drosophila and cultured cell studies supports a role for heterotrimeric guanosine triphosphate-binding proteins (G proteins) in Wnt signaling. Wnt inhibits the degradation of the transcriptional regulator beta-catenin. We screened the alpha and betagamma subunits of major families of G proteins in a Xenopus egg extract system that reconstitutes beta-catenin degradation. We found that Galpha(o), Galpha(q), Galpha(i2), and Gbetagamma inhibited beta-catenin degradation. Gbeta(1)gamma(2) promoted the phosphorylation and activation of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) by recruiting glycogen synthase kinase 3 (GSK3) to the membrane and enhancing its kinase activity. In both a reporter gene assay and an in vivo assay, c-betaARK (C-terminal domain of beta-adrenergic receptor kinase), an inhibitor of Gbetagamma, blocked LRP6 activity. Several components of the Wnt-beta-catenin pathway formed a complex: Gbeta(1)gamma(2), LRP6, GSK3, axin, and dishevelled. We propose that free Gbetagamma and Galpha subunits, released from activated G proteins, act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription.

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.

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., (2009 Elsevier Ltd. All rights reserved.)

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.

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.

G protein-coupled receptor kinase 2 (GRK2) modulates G protein-coupled receptor desensitization and signaling. We previously described down-regulation of GRK2 expression in vivo in rat neonatal brain following hypoxia-ischemia. In this study, we investigated the molecular mechanisms involved in GRK2 down-regulation, using organotypic cultures of neonatal rat hippocampal slices exposed to oxygen and glucose deprivation (OGD). We observed a 40% decrease in GRK2 expression 4 h post-OGD. No changes in GRK2 protein occurred after exposure of hippocampal slices to glucose deprivation only. No significant alterations in GRK2 mRNA expression were detected, suggesting a post-transcriptional effect of OGD on GRK2 expression. Blockade of the proteasome pathway by MG132 prevented OGD-induced decrease of GRK2. It has been shown that extracellular signal-regulated kinase-dependent phosphorylation of GRK2 at Ser670 triggers its turnover via the proteasome pathway. However, despite a significant increase of pSer670-GRK2 after OGD, inhibition of the extracellular signal-regulated kinase pathway by PD98059 did neither prevent the hypoxia-ischemia-induced increase in pSer670-GRK2 nor the down-regulation of GRK2 protein. Interestingly, inhibition of phosphoinositide-3-kinase with wortmannin inhibits both OGD-induced phosphorylation of GRK2 on Ser670 and the GRK2 decrease. In conclusion, OGD-induced phosphoinositide-3-kinase-dependent phosphorylation of GRK2 on Ser670 is a novel mechanism leading to down-regulation of GRK2 protein via a proteasome-dependent pathway.

The G protein-coupled receptor kinase 3 gene (GRK3) is a candidate gene for cocaine addiction because it is involved in the regulation of several neurotransmitter receptors, including the response to dopaminergic agonists such as methamphetamine and cocaine. We hypothesized that genetic variants in the GRK3 gene might be associated with an increased risk of cocaine addiction. To test this, we genotyped three variants located in 5' untranslated and promoter regions of the gene in a sample of 711 cocaine users and 862 healthy control individuals from Sao Paulo, Brazil. Genotypic, allelic and haplotypic analyses provided no evidence for an association between alleles at these polymorphisms and cocaine abuse in this sample. Population stratification was tested for and its effect corrected for, but this did not affect the association test results. In conclusion, our results do not support a major role for GRK3 gene promoter variants in cocaine addiction.

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.

G-protein coupled receptor kinase 3 (GRK3) mediates desensitization of alpha(2)-adrenergic (alpha(2)-AR) and CRF(1) receptors. CRF(1) receptors, alpha(2)-AR and GRK3, are localized to the primary source of noradrenergic inputs to higher brain centers critical in both the response to stress and the development of depression, namely, locus coeruleus (LC). This study utilizing CATH.a cells (derived from the LC), demonstrates for the first time, that the stress hormone, CRF selectively up-regulates GRK3 expression via an ERK1/2-mediated mechanism accompanied by the activation of Sp-1 and Ap-2 transcription factors. This observation has important implications for the regulation of stress signaling in the brain.

G protein-coupled receptors represent the most diverse group of proteins involved in transmembrane signalling, that participate in the regulation of a wide range of physicochemical messengers through the interaction with heterotrimeric G proteins. In addition, GPCRs stimulation also triggers a negative feedback mechanism, known as desensitization that prevents the potentially harmful effects caused by persistent receptor stimulation. In this adaptative response, G protein-coupled receptor kinases (GRKs) play a key role and alterations in their function are related to diverse pathophysiological situations. Based on the scarce knowledge about the regulation of GRK2 by other kinases of the same family, the aim of the present work was to investigate the regulation of GRK2 levels in systems where other GRKs are diminished by antisense technique. Present findings show that in U937 cells GRK2 levels are regulated by GRK3 and not by GRK6 through a mechanism involving InsP upregulation. This work reports a novel GRK3-mediated GRK2 regulatory mechanism and further suggests that GRK2 may also act as a compensatory kinase tending to counterbalance the reduction in GRK3 levels. This study provides the first evidence for the existence of GRKs cross-regulation.

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.

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.

The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca(2+)(e)) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to beta-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and beta-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca(2+)(e)-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in G alpha(q) binding (D110A) was without major effect. Overexpression of GRK 4-6 did not reduce Ca(2+)(e)-dependent inositol phosphate accumulation. Overexpression of beta-arrestin 1 or 2 revealed a modest inhibitory effect on Ca(2+)(e)-dependent inositol phosphate production (20-30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10-15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to beta-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to G alpha(q).

In the heart, ACh activates the ACh-activated K(+) current (I(K,ACh)) via the M(2) muscarinic receptor. The relationship between desensitization of I(K,ACh) and internalization of the M(2) receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, I(K,ACh) declined by approximately 62% with time constants of 1.5 and 26.9 min, whereas approximately 83% of the M(2) receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with beta-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and beta-arrestin 2 significantly increased I(K,ACh) desensitization and M(2) receptor internalization during a 3-min application of agonist. Internalized M(2) receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and beta-arrestin 2-dependent internalization of the M(2) receptor into clathrin-coated vesicles could play a major role in I(K,ACh) desensitization.

Relatively small changes in G-protein-coupled receptor kinase (GRK) 3 expression (approximately 2-fold) profoundly affect alpha2-adrenergic receptor (AR) function and preferentially regulate neuronal alpha2A- and alpha2B-AR signaling. In the present study, we provide evidence that epinephrine (EPI)-induced up-regulation of GRK3 protein expression in two neuronal cell lines, BE(2)-C cells (endogenously express alpha2A- and beta2AR) and BN17 cells [endogenously express alpha2B (NG108) and transfected to express beta2-AR] is due in part to increased GRK3 gene expression. In both cell lines, the increase in GRK3 transcription occurred via an extracellular signal-regulated kinase (ERK) 1/2-dependent mechanism because the increase in GRK3 mRNA is eliminated in the presence of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2-amino phenylthiobutadiene)]. EPI-induced GRK3 mRNA up-regulation also is prevented in the presence of propranolol or phentolamine. Moreover, GRK3 mRNA did not increase in response to EPI treatment in NG108 cells (endogenously express alpha2B-AR with no beta2-AR). Both these results suggest that simultaneous activation of alpha2- and beta2-AR by EPI is required for the ERK1/2-dependent increase in GRK3 mRNA. The EPI-induced increase in GRK3 mRNA was unaffected in the presence of the protein kinase C inhibitor, chelerythrine chloride. Finally, EPI treatment resulted in increased nuclear translocation and accumulation of the transcription factors, Sp-1 and Ap-2, in BE(2)-C cells. Taken together, our results demonstrate the involvement of the ERK1/2 pathway in selective up-regulation of GRK3 mRNA expression, possibly via activation of Sp-1 and Ap-2 transcription factors in neuronal cells.

Alterations in signal transduction pathway of G-protein-coupled receptors (GPCRs) have been found in the cerebrocortex and in the peripheral cultured tissues of patients with Alzheimer's disease (AD). The G-protein-coupled receptor kinase-2 (GRK2) plays an important role in regulating the GPCRs signaling: its increased expression is associated with receptor desensitization. The aim of this study was to explore GRK2 levels in peripheral lymphocytes of AD patients and to establish a correlation between lymphocyte protein concentrations and the degree of cognitive impairment. GRK2 mRNA and protein expression were evaluated in the lymphocytes of AD patients with mild or moderate/severe cognitive impairment and in age-matched healthy subjects. Both GRK2 mRNA and protein expression were higher in AD patients lymphocytes compared to controls. Furthermore, lymphocyte GRK2 levels were significantly correlated to the degree of cognitive decline. Our preliminary data suggest that GRK2 is involved in GPCRs coupling dysfunction observed in AD patients. Further studies are needed in order to verify whether the lymphocyte GRK2 might be utilized as a novel biomarker in AD diagnosis and clinical monitoring.

G protein-coupled receptor kinase 2 (GRK2) is involved in the agonist-induced desensitization of beta2-adrenoceptors. In addition, GRK2 is capable of binding and phosphorylating tubulin. Interestingly, microtubule dynamics profoundly affect agonist-induced internalization of beta2-adrenoceptors. Here, we analyzed agonist-induced beta2-adrenoceptor internalization and signaling in splenocytes from GRK2+/- mice that have a approximately 50% lower level of GRK2 protein compared to wild type (WT) mice. In addition, we investigated the role of microtubule stability in these processes. Splenocytes from GRK2+/- mice express approximately 50% less beta2-adrenoceptors on the cell surface and show impaired agonist-induced beta2-adrenoceptor internalization. Disruption of microtubules using colchicine reduces agonist-induced beta2-adrenoceptor internalization in cells from WT, but not in cells from GRK2+/- mice. Importantly, increasing tubulin stability by taxol almost completely restores the defective agonist-induced beta2-adrenoceptor internalization in cells from GRK2+/- animals, without affecting WT cells. Despite lower surface receptor numbers, cells of GRK2+/- mice show normal beta2-adrenoceptor agonist-induced cAMP responses. Although interfering with microtubule stability has major effects on agonist-induced receptor internalization in GRK2+/- cells, microtubule dynamics do not influence cAMP responses. Our data suggest that cells with low GRK2 adapt to the lower GRK2 level by decreasing the number of beta2-adrenoceptors on the cell surface. In addition, the cellular GRK2 level determines the extent of agonist-induced beta2-adrenoceptor internalization via a mechanism involving microtubule stability. Importantly, however, normalization of agonist-induced receptor internalization by taxol is not sufficient to alter receptor signaling.

To identify candidate genes relevant for prostate tumour prognosis and progression, we performed an exhaustive gene search in seven previously described genomic-profiling studies of 161 prostate tumours, and four expression profiling studies of 61 tumours. From the resulting list of candidate genes, six were selected for protein-expression analysis based on the availability of antibodies applicable to paraffinised tissue: fatty acid synthase (FASN), MYC, beta-adrenergic receptor kinase 1 (BARK1, GRK2) the catalytic subunits of protein phosphatases PP1alpha (PPP1CA) and PP2A (PPP2CB) and metastasis suppressor NM23-H1. These candidates were analysed by immunohistochemistry (IHC) on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias (BPH) from 175 patients. In univariate analysis, expression of PP1alpha (P=0.001) was found to strongly correlate with Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score (P<0.001 each) in univariate as well as in multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterised by a complete loss of BARK1 protein (P=0.023). In conclusion, our study revealed novel molecular markers of potential diagnostic and therapeutic relevance for prostate carcinoma.

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.

Transgenic mice with cardiac specific overexpression of beta-adrenergic receptor kinase-1 (betaARK-1) exhibit reduced contractility in the presence of adrenergic stimulation. However, whether contractility is altered in the absence of exogenous agonist is not clear. Effects of betaARK-1 overexpression on contraction were examined in mouse ventricular myocytes, studied at 37 degrees C, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microelectrodes (18-26 MOmega; 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in betaARK-1 cells than in wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1-3 MOmega; 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl(2), 2.5 mM KH(2)PO(4), 0.12 mM CaCl(2), 0.5 mM EGTA, and 10 mM HEPES), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. Furthermore, when cells were dialyzed with solutions that contained phosphodiesterase-sensitive sodium-cAMP (50 microM), the extent of cell shortening was similar in wild-type and betaARK-1 myocytes. However, when patch solutions were supplemented with phosphodiesterase-resistant 8-bromo-cAMP (50 muM), contractions were larger in betaARK-1 than wild-type cells. This difference was eliminated by the protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). Interestingly, Ca(2+) current amplitudes and inactivation rates were similar in betaARK-1 and wild-type cells in all experiments. These results suggest components of the adenylyl cyclase-protein kinase A pathway are sensitized by chronically increased betaARK-1 activity, which may augment contractile function in the absence of exogenous agonist. Thus, changes in contractile function in myocytes from failing hearts may reflect, in part, effects of chronic up-regulation of betaARK-1 on the cAMP-protein kinase A pathway.

G-protein coupled receptor kinases (GRKs) constitute a serine/threonine kinase family playing a major role in agonist-induced phosphorylation and desensitization of G-protein coupled receptors. Recently, GRK2 and GRK5 have been demonstrated to phosphorylate alpha-synuclein (Ser129) and other synuclein isoforms. We studied colocalization of GRK2, GRK5, alpha-synuclein, and tau in neurodegenerative disorders characterized by fibrillary tau inclusions and/or alpha-synuclein-enriched Lewy bodies. We found that Lewy bodies were negative for both GRK2 and GRK5 in Lewy body disease (LBD) and LBD mixed with Alzheimer disease (AD + LBD). Instead, GRK2 but not GRK5 colocalized with 40% to 50% of neurofibrillary tangles in AD + LBD and AD brains. In disorders with less prominent alpha-synucleinopathy, neuronal and glial fibrillary tau deposits known to contain distinct subsets of tau isoforms were also positive for GRK2. These deposits included tufted astrocytes and coiled bodies in progressive supranuclear palsy, astrocytic plaques in corticobasal degeneration, and Pick bodies in Pick disease. In addition, paired helical filaments isolated from AD and AD + LBD brains were found to immunogold-label for GRK2, suggesting that GRK2 could be a potential tau kinase associated with fibrillary tau. Our studies indicate that GRK2 is a novel component of neuronal and glial fibrillary tau deposits with no preference in tau isoform binding. GRK2 may play a role in hyperphosphorylation of tau in tauopathies.

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

In this study we investigated the mechanisms responsible for MAP kinase ERK1/2 activation following agonist activation of endogenous mu opioid receptors (MOR) normally expressed in cultured striatal neurons. Treatment with the MOR agonist fentanyl caused significant activation of ERK1/2 in neurons derived from wild type mice. Fentanyl effects were blocked by the opioid antagonist naloxone and were not evident in neurons derived from MOR knock-out (-/-) mice. In contrast, ERK1/2 activation by fentanyl was not evident in neurons from GRK3-/- mice or neurons pretreated with small inhibitory RNA for arrestin3. Consistent with this observation, treatment with the opiate morphine (which is less able to activate arrestin) did not elicit ERK1/2 activation in wild type neurons; however, transfection of arrestin3-(R170E) (a dominant positive form of arrestin that does not require receptor phosphorylation for activation) enabled morphine activation of ERK1/2. In addition, activation of ERK1/2 by fentanyl and morphine was rescued in GRK3-/- neurons following transfection with dominant positive arrestin3-(R170E). The activation of ERK1/2 appeared to be selective as p38 MAP kinase activation was not increased by either fentanyl or morphine treatment in neurons from wild type, MOR-/-, or GRK3-/- mice. In addition, U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) blocked ERK1/2 activation by fentanyl. These results support the hypothesis that MOR activation of ERK1/2 requires opioid receptor phosphorylation by GRK3 and association of arrestin3 to initiate the cascade resulting in ERK1/2 phosphorylation in striatal neurons.

The deregulation of inflammatory response during sepsis seems to reflect the overproduction of mediators, which suppress leukocyte functions. We investigated the intracellular mechanisms underlying the inability of neutrophils from severe septic patients to migrate toward chemoattractants. Patients with sepsis (52) and 15 volunteers were prospectively enrolled. Patients presented increased circulating levels of tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-8, and IL-10. Patients showed reduced neutrophil chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or IL-8. No difference in the transcription or expression of the IL-8 receptor, CXCR1, was detected in neutrophils from controls and patients. However, septic neutrophils failed to increase tyrosine phosphorylation and actin polymerization in response to IL-8 or LTB4. In contrast, septic neutrophils, similar to controls, showed phagocytic activity that induced actin polymerization and augmented phosphotyrosine content. Treatment of control neutrophils with cytokines and lipopolysaccharide (LPS) to mimic endogenous septic environment inhibited actin polymerization and tyrosine phosphorylation in response to IL-8 or LTB4. High expression of G protein-coupled receptor kinase 2 (GRK2) and GRK5 was detected in septic neutrophils and control cells treated with cytokines plus LPS. Data suggest that endogenous mediators produced during sepsis might continually activate circulating neutrophils, leading to GRK activation, which may induce neutrophil desensitization to chemoattractants.

G-protein receptor kinase 2 (GRK2) is 1 of 7 mammalian GRKs that phosphorylate ligand-bound 7-transmembrane receptors, causing receptor uncoupling from G proteins and potentially activating non-G-protein signaling pathways. GRK2 is unique among members of the GRK family in that its genetic ablation causes embryonic lethality. Cardiac abnormalities in GRK2 null embryos implicated GRK2 in cardiac development but prevented studies of the knockout phenotype in adult hearts. Here, we created GRK2-loxP-targeted mice and used Cre recombination to generate germline and cardiac-specific GRK2 knockouts. GRK2 deletion in the preimplantation embryo with EIIa-Cre (germline null) resulted in developmental retardation and embryonic lethality between embryonic day 10.5 (E10.5) and E11.5. At E9.5, cardiac myocyte specification and cardiac looping were normal, but ventricular development was delayed. Cardiomyocyte-specific ablation of GRK2 in the embryo with Nkx2.5-driven Cre (cardiac-specific GRK2 knockout) produced viable mice with normal heart structure, function, and cardiac gene expression. Cardiac-specific GRK2 knockout mice exhibited enhanced inotropic sensitivity to the beta-adrenergic receptor agonist isoproterenol, with impairment of normal inotropic and lusitropic tachyphylaxis, and exhibited accelerated development of catecholamine toxicity with chronic isoproterenol treatment. These findings show that cardiomyocyte autonomous GRK2 is not essential for myocardial development after cardiac specification, suggesting that embryonic developmental abnormalities may be attributable to extracardiac effects of GRK2 ablation. In the adult heart, cardiac GRK2 is a major factor regulating inotropic and lusitropic tachyphylaxis to beta-adrenergic agonist, which likely contributes to its protective effects in catecholamine cardiomyopathy.

Adverse remodeling after myocardial infarction (MI) determines the progression of heart failure. Failing hearts are characterized by downregulation of beta-adrenergic receptor (beta-AR) signaling in part because of increased beta-AR kinase 1 activity. Our previous studies have shown that overexpression of the phosphoinositide kinase (PIK) domain of phosphoinositide 3-kinase (PI3K), prevents beta-AR downregulation and enhances adrenergic agonist responsiveness by inhibiting the targeting of PI3K to the beta-AR complex. To investigate whether preventing beta-AR downregulation in the heart ameliorates cardiac function post-MI, transgenic mice with cardiac-specific overexpression of the PIK domain peptide (TgPIK) underwent left coronary artery ligation and were subsequently followed by serial echocardiography at 4, 8, 12, 16, and 20 wk. Despite having similar infarction sizes, TgPIK mice showed better systolic function, less cardiac dilatation, and improved hemodynamic response to dobutamine compared with littermate controls after MI. To test that displacement of PI3K from the beta-AR complex, but not the total loss of PI3K-gamma, is critical for amelioration of cardiac function, mice lacking the PI3K-gamma (PI3K-gamma-KO) underwent MI, and their cardiac function was assessed 20 wk post-MI. Serial echocardiographic measurements showed severe reduction in contractile performance in PI3K-gamma-KO compared with TgPIK mice. Furthermore, significant beta-AR downregulation and desensitization were only seen in infarcted wild-type and PI3K-gamma-KO mice and not in TgPIK mice. Together, these results demonstrate that adverse remodeling of the ventricle after MI can be attenuated by a strategy that prevents recruitment of PI3K to the plasma membrane and restores normal beta-AR function.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

Aim: We examine the expression of mRNA of G protein-coupled receptor kinase (GRK) subtypes and muscarinic acetylcholine receptor (M) subtypes in the detrusor smooth muscle of the human urinary bladder. Furthermore, we confirm the presence and the localization of GRK proteins in the detrusor smooth muscle of the obstructed bladder in comparison with the control bladder., Methods: Detrusor smooth muscle tissues of the human urinary bladder were obtained from 12 male patients; 6 patients did not have bladder outlet obstruction, and the other 6 patients had bladder outlet obstruction. Portions of the dome or anterior wall of the urinary bladder were used for the present study. Reverse transcription/polymerase chain reaction for GRK2, M2 and M3 was performed using total RNA extracted from human urinary bladder detrusor. Antibodies to GRK2, GRK3 and GRK4 were used to confirm the presence of the protein product in the human urinary bladder using immunohistochemical staining and the western blotting technique., Results: All complementary DNA (cDNA) transcribed from three different mRNA (M2, M3 and GRK2) were successfully amplified and size-fractionated. The expression of GRK2 protein was strong in the human bladder detrusor, but was significantly weakened by western blotting in obstructed bladder in comparison with control bladder., Conclusions: Failure in desensitization mechanisms of muscarinic acetylcholine receptors might be related to storage symptom elicited by overactivity in obstructed bladder with benign prostatic hyperplasia.

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that are known to contribute to the adaptation of the heptahelical G protein-coupled receptors (GPCRs) and to regulate downstream signals through these receptors. GPCRs mediate the action of messengers that are key modulators of cardiac and vascular cell function, such as growth and differentiation. GRKs are members of a multigene family, which are classified into three subfamilies and are found in cardiac, vascular and cerebral tissues. Increasing evidence strongly supports the hypothesis that vascular damage is an early contributor to the development of Alzheimer disease (AD) and/or other pathology that can mimic human AD. Based on this hypothesis, and since kinases of this family are known to regulate numerous receptor functions both in the brain, myocardium and elsewhere, we explored cellular and subcellular localization by immunoreactivity of G protein-coupled receptor kinase 2 (GRK2), also known as beta-adrenergic receptor kinase-1(betaARK1), in the early pathogenesis of AD and in ischemia reperfusion injury models of brain hypoperfusion. In the present study, we used the two-vessel carotid artery occlusion model, namely the 2-VO system that results in chronic brain hypoperfusion (CBH) and mimics mild cognitive impairment (MCI) and vascular changes in AD pathology. Our findings demonstrate the early overexpression of GRK2 member kinase in the cerebrovasculature, especially endothelial cells (EC) following CBH, as well as in select cells from human AD tissue. We found a significant increase in GRK2 immunoreactivity in the EC of AD patients and after CBH, which preceded any amyloid deposition. Since GRK2 activity is associated with certain compensatory changes in brain cellular compartments and in ischemic cardiac tissue, our findings suggest that chronic hypoperfusion initiates oxidative stress in these conditions and appears to be the main initiating injury stimulus for disruption of brain and cerebrovascular homeostasis and metabolism.

The effect of exercise on beta-adrenergic receptor (beta-AR) trafficking was investigated in rat adipocytes. The binding sites of a hydrophilic ligand, [(3)H]CGP12177, increased immediately (0 h) and at 3 h after exercise (3 h) but decreased at 24 h after exercise (24 h). The data of immunoblotting revealed that the alterations in the binding sites mainly paralleled the alterations in the beta2-AR proteins in membrane fractions. The protein expressions of both G-protein-coupled receptor kinase (GRK)-2 and beta-arrestin-2 were reduced, with a decline in beta2-AR ubiquitination at 0 h and 3 h. The protein expressions of beta2-AR, GRK-2, beta-arrestin-2, the beta2-AR/beta-arrestin-2 complex, and beta2-AR ubiquitination returned to their respective control levels at 24 h, whereas the beta2-AR mRNA level was reduced. Administration of either lactacystin or propranolol did not alter GRK-2 and beta2-AR protein expressions after exercise. Thus, the mechanism underlying the increased density of beta2-AR up to at least 3 h may involve alterations in a multistep event involving the coordinate interaction among proteins mediating beta2-AR trafficking, in which both the receptor-agonist interactions and ubiquitin-proteasome pathway have a key role. However, the decreased protein expression of beta2-AR at 24 h might be due to some change occurring at the translational levels.

Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca(2+) calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, beta-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells produced a significant reduction of the extracellular signal-regulated kinase (ERK) response to CCL2. This effect is independent of its role in receptor phosphorylation because the kinase-deficient mutant GRK2K220R was able to reduce this response, and ERK activation by CCR2BIX, a phosphorylation-defective receptor mutant, was also inhibited by GRK2. Constructs containing the Galpha(q)-binding RGS-like RH domain of GRK2 or its Gbetagamma-binding domain could not reproduce the inhibition, thus revealing that GRK2 acts downstream of G proteins. Interestingly, chemokine-driven mitogen-activated protein kinase kinase (MEK) stimulation is not affected in cells overexpressing GRK2 or GRK2K220R or in splenocytes from heterozygous GRK2 mice, where reduced kinase levels correlate with enhanced ERK activation by chemokines. We find GRK2 and MEK in the same multimolecular complex, thus suggesting a mechanism for GRK2 regulation of ERK activity that involves a direct or coordinate interaction with MEK. These results suggest an important role for GRK2 in the control of chemokine induction of ERK activation at the level of the MEK-ERK interface.

Purkinje cell protein-2 (Pcp2 or L7) is highly expressed in cerebellar Purkinje cells and retinal bipolar neurons and interacts with the Galpha(i/o) family of G-proteins. Although the expression pattern of Pcp2 in the developing central nervous system suggests a role in differentiation, its function remains unknown. We established Tet-off inducible expression of Pcp2 in PC12 cells (rat pheochromocytoma cells) to determine whether Pcp2 regulates neuronal differentiation. Utilizing a polyclonal antibody, Pcp2 was localized in the cell body and throughout neurites of differentiated PC12 cells, similar to its localization in cerebellar Purkinje cells. Pcp2 expression in PC12 cells stimulated process formation (5-fold) and NGF (nerve growth factor)-stimulated neurite length (2-fold). Under basal conditions, Pcp2-PC12 cells demonstrated a 5-fold increase in Ras activation relative to non-induced PC12 cells and there was no change in extracellular-signal-regulated kinase 1/2 activity with Pcp2 expression. However, Pcp2 induction led to a >3-fold increase in basal p38 MAPK (mitogen-activated protein kinase) activity and the addition of NGF significantly stimulated both Ras and p38 MAPK in Pcp2-PC12 cells relative to the controls. Pretreatment of Pcp2-PC12 cells with the p38-specific inhibitor SB203580 blocked both the increased neurite formation and NGF-stimulated neurite growth. Pertussis toxin treatment had no effect on neurite growth in control cells, but completely blocked Pcp2-mediated increased neurite growth. Transient transfection of the beta-adrenergic receptor kinase C-terminus to prevent signalling through Gbetagamma in Pcp2-PC12 cells also inhibited the Pcp2-induced phenotype and reduced the Pcp2-stimulated Ras activation. Taken together, these findings demonstrate that Pcp2 induces differentiation in PC12 cells, in part through Gbetagamma-mediated Ras and p38 MAPK activation and suggest the potential for similar signalling mechanisms in Purkinje cells.

We have previously demonstrated that concomitant activation of receptor tyrosine kinases and certain G protein-coupled receptors (GPCRs) can promote a synergistic increase in the rate of airway smooth muscle cell (ASM) proliferation. Here we clarify the role of p70S6 kinase (p70S6K) as an integrator of receptor tyrosine kinase and GPCR signaling that augments ASM DNA synthesis by demonstrating that specific p70S6K phosphorylation sites receive distinct regulatory input from GPCRs that promotes sustained kinase activity critical to mitogenesis. Prolonged stimulation of ASM cells with EGF and thrombin induced a greater than additive effect in levels of p70S6K phosphorylated at residue T389, whereas a significant but more modest increase in the level of T229 and T421/S424 phosphorylation was also observed. The augmenting effects of thrombin could be dissociated from p42/p44 MAPK activation, as selective inhibition of thrombin-stimulated p42/p44 failed to alter the profile of cooperative p70S6K T389 phosphorylation, p70S6K kinase activity, or ASM [(3)H]thymidine incorporation. Thrombin stimulated a sustained increase in the level of Akt phosphorylation and also augmented EGF-stimulated Akt phosphorylation. The cooperative effects of thrombin on Akt/p70S6K phosphorylation and [(3)H]thymidine incorporation were all attenuated by heterologous expression of Gbetagamma sequestrants. These data suggest that PI3K-dependent T389/T229 phosphorylation is limiting in late-phase p70S6K activation by EGF and contributes to the cooperative effect of GPCRs on p70S6K activity and cell growth.

G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.

Background: Ischemic preconditioning (IPC) protects hearts against ischemia by reducing infarct size. However, IPC does not preserve cardiac function, such as left ventricular peak developed pressure (LVPDP). Moreover, IPC fails to protect the post-myocardial infarct (MI) heart., Design: Rat hearts were transfected with beta2-adrenergic receptor (B2AR) cDNA by the hemagglutinating virus of Japan-liposome method. After the gene transfer, the hearts were perfused in a Langendorff mode and preconditioned with two cycles of 5 min of ischemia and reperfusion. After 20 min of global ischemia, the hearts were reperfused under aerobic conditions for 90 min. LVPDP was measured as an indicator of the cardiac function., Results: LVPDP of ischemic hearts was well preserved by the combination treatment of IPC and gene transfer of B2AR, but not IPC or gene transfer of B2AR alone. Moreover, the treatment was beneficial to even the post-MI heart. On the contrary, gene transfer of beta-adrenergic receptor kinase 1 (BARK1) reduced the protective effect of IPC. We also found that the mRNA ratio of B2AR and BARK1 was well correlated with the preservation of the LVPDP., Conclusions: The combination treatment of IPC and gene transfer of B2AR protects cardiac function against ischemia and it shows the beneficial effect also in post-MI hearts.

Stimulation of the histamine H1 receptor has been shown to enhance adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation in various cell types but, to date, the mechanism by which this occurs is still unclear. In the present study, we examined the possibility that the betagamma subunits of G proteins (G betagamma) are involved in this process in cultured Chinese hamster ovary cells transfected with the human histamine H1 receptor (CHO-H1). Histamine increased intracellular cAMP levels in a concentration-dependent manner in CHO-H1 cells, and this histamine action was abolished by pyrilamine (1 microM). Inhibition of histamine H1 receptor-G(q) protein coupling by stable expression of the C-terminal peptide of G alpha(q) protein significantly attenuated the cAMP accumulation induced by histamine. By comparison, neither BAPTA/AM (50 microM), an intracellular Ca2+ chelator, nor GF 109203X (1 microM), an inhibitor of protein kinase C, influenced the cAMP response. Histamine H1 receptor-mediated cAMP accumulation was significantly inhibited by transient transfection of CHO-H1 cells with the C-terminal peptide of beta-adrenoceptor kinase I (residues 542-685), a scavenger of G betagamma. Stable expression of the C-terminal peptide of the G alpha(s) protein, but not treatment with pertussis toxin (200 ng/ml for 24 h), attenuated the histamine H1 receptor-mediated cAMP accumulation. These results suggest that stimulation of histamine H1 receptors activates adenylyl cyclase through the release of G betagamma subunits from G proteins, thereby elevating intracellular cAMP levels.