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Schistosomes are blood-dwelling trematodes with global impact on human and animal health. Because medical treatment is currently based on a single drug, praziquantel, there is urgent need for the development of alternative control strategies. The Schistosoma mansoni genome project provides a platform to study and connect the genetic repertoire of schistosomes to specific biological functions essential for successful parasitism. G protein–coupled receptors (GPCRs) form the largest superfamily of transmembrane receptors throughout the Eumetazoan phyla, including platyhelminths. Due to their involvement in diverse biological processes, their pharmacological importance, and proven druggability, GPCRs are promising targets for new anthelmintics. However, to identify candidate receptors, a more detailed understanding of the roles of GPCR signalling in schistosome biology is essential. An updated phylogenetic analysis of the S. mansoni GPCR genome (GPCRome) is presented, facilitated by updated genome data that allowed a more precise annotation of GPCRs. Additionally, we review the current knowledge on GPCR signalling in this parasite and provide new insights into the potential roles of GPCRs in schistosome reproduction based on the findings of a recent tissue-specific transcriptomic study in paired and unpaired S. mansoni. According to the current analysis, GPCRs contribute to gonad-specific functions but also to nongonad, pairing-dependent processes. The latter may regulate gonad-unrelated functions during the multifaceted male–female interaction. Finally, we compare the schistosome GPCRome to that of another parasitic trematode, Fasciola, and discuss the importance of GPCRs to basic and applied research. Phylogenetic analyses display GPCR diversity in free-living and parasitic platyhelminths and suggest diverse functions in schistosomes. Although their roles need to be substantiated by functional studies in the future, the data support the selection of GPCR candidates for basic and applied studies, invigorating the exploitation of this important receptor class for drug discovery against schistosomes but also other trematodes.

Salmeterol is a long-acting β2-adrenergic receptor (β2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the β2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein-coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded-β2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to β2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRETISO and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein-coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A-mediated events.

G protein-coupled receptor kinase 6 (GRK6) plays a universal role in receptor desensitization, by acting as a receptor-G protein interface, thereby affecting serine/threonine kinases. In this study, a 20-aa-long peptide of human GRK6 C-terminus domain was synthesized and covalently coupled to keyhole limpet hemocyanin (KLH). A mouse monoclonal antibody against human GRK6 (anti-GRK6 MAb) was successfully prepared through hybridoma technique by immunizing BALB/c mice with synthesized GRK6426-446-KLH peptides. A high specificity and affinity strain of hybridoma 5D12 were established. The titer of the purified anti-GRK6 MAb was 1.28 × 10(6) measured by indirect ELISA. Western blot and immunocytochemistry experiments were also applied to characterize the antibody specificity. Antibody absorption assays showed that the anti-GRK6 MAb can be blocked by GRK6426-446 peptides. These results indicated that the antibody could bind to GRK6 antigen specifically. This MAb provides valuable support for further studies on the functional properties of GRK6.

INTRODUCTION AND OBJECTIVES Pulmonary regurgitation (PR) is a frequent complication after repair of congenital heart disease. Lymphocyte expression of adrenoceptors (β1 and β2) and kinases (GRK2, GRK3, and GRK5) reflects the neurohumoral changes that occur in heart failure (HF). The main objective of this study was to describe the gene expression of these molecules in circulating lymphocytes in patients with severe PR. METHODS A prospective study was conducted to analyze lymphocyte expression of these molecules in patients with severe PR and compare it with expression in healthy controls and patients with advanced HF. RESULTS We studied 35 patients with severe PR, 22 healthy controls, and 13 patients with HF. Multiple comparisons analysis showed that β2-adrenoceptor gene expression levels were higher in the control group than in patients in the PR and HF groups and that expression in the latter 2 groups was similar (748.49 [rank 1703.87] vs 402.80 [rank 1210.81] vs 287.46 [rank 685.69] P = .001). Similar findings were obtained in gene expression of GRK2 (760.89 [rank 1169.46] vs 445.17 [rank 1190.69] vs 284.09 [rank 585.27] P < .001). There were no differences in expression levels of these molecules according to clinical variables in patients with PR. CONCLUSIONS The gene expression pattern of GRK2 and β2-adrenoceptor as molecular markers of cardiac dysfunction was altered in patients with severe PR compared with controls and was similar to expression in patients with advanced HF.

Different isoforms of the genes involved in phototransduction are expressed in vertebrate rod and cone photoreceptors, providing a unique example of parallel evolution via gene duplication. In this study, we determine the molecular phylogeny of the proteins underlying the shut-off steps of phototransduction in the agnathan and jawed vertebrate lineages. For the G-protein receptor kinases (GRKs), the GRK1 and GRK7 divisions arose prior to the divergence of tunicates, with further expansion during the two rounds of whole-genome duplication (2R); subsequently, jawed and agnathan vertebrates retained different subsets of three isoforms of GRK. For the arrestins, gene expansion occurred during 2R. Importantly, both for GRKs and arrestins, the respective rod isoforms did not emerge until the second round of 2R, just prior to the separation of jawed and agnathan vertebrates. For the triplet of proteins mediating shut-off of the G-protein transducin, RGS9 diverged from RGS11, probably at the second round of 2R, whereas Gβ5 and R9AP appear not to have undergone 2R expansion. Overall, our analysis provides a description of the duplications and losses of phototransduction shut-off genes that occurred during the transition from a chordate with only cone-like photoreceptors to an ancestral vertebrate with both cone- and rod-like photoreceptors.

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. Emerging evidence demonstrates that signaling through GPCRs affects numerous aspects of cancer biology such as vascular remolding, invasion, and migration. Therefore, development of GPCR-targeted drugs could provide a new therapeutic strategy to treating a variety of cancers. G protein-coupled receptor kinases (GRKs) modulate GPCR signaling by interacting with the ligand-activated GPCR and phosphorylating its intracellular domain. This phosphorylation initiates receptor desensitization and internalization, which inhibits downstream signaling pathways related to cancer progression. GRKs can also regulate non-GPCR substrates, resulting in the modulation of a different set of pathophysiological pathways. In this review, we will discuss the role of GRKs in modulating cell signaling and cancer progression, as well as the therapeutic potential of targeting GRKs.

G protein-coupled receptor kinases (GRKs) are classically known for their role in regulating the activity of the largest known class of membrane receptors, which influence diverse biological processes in every cell type in the human body. As researchers have tried to uncover how this family of kinases, containing only 7 members, achieves selective and coordinated control of receptors, they have uncovered a growing number of non-canonical activities for these kinases. These activities include phosphorylation of non-receptor targets and kinase-independent molecular interactions. In particular, GRK2, GRK3, and GRK5 are the predominant members expressed in the heart. Their canonical and non-canonical actions within cardiac and other tissues have significant implications for cardiovascular function in healthy animals and for the development and progression of disease. This review summarizes what is currently known regarding the activity of these kinases, and particularly the role of GRK2 and GRK5 in the molecular alterations that occur during heart failure. This review further highlights areas of GRK regulation that remain poorly understood and how they may represent novel targets for therapeutic development.

Cell-cell communication is essential for plants to integrate developmental programs with external cues that affect their growth. Recent advances in plant signaling have uncovered similar molecular mechanisms in shoot, root, and vascular meristem signaling that involve receptor-like kinases and small, secreted peptides. Here, we report that the receptor-like kinases TOAD2/RPK2 and RPK1 regulate root growth by controlling cell proliferation and affecting meristem size. Two types of developmental alterations were observed upon exogenous CLE peptide application. The first type was detected in all plants treated, and comprise increased proliferative activity of cells in the stem cell niche and a delay of progression in differentiation of daughter cells. The second type was changes specific to the genotypes that are sensitive to CLE-driven root meristem inhibition and include a large decrease in the occurrence of cell divisions in longitudinal files, correlating with shorter meristems and cessation of root growth. The root meristems of

G protein-coupled receptor kinase-6 (GRK6) is a serine/threonine kinase that is important in inflammatory processes. In this study, we examined the role of GRK6 in Escherichia coli-induced lung infection and inflammation using GRK6 knockout (KO) and wild-type (WT) mice. Intratracheal instillation of E. coli significantly enhanced bacterial load in the bronchoalveolar lavage (BAL) of KO compared with WT mice. Reduced bacterial clearance in the KO mice was not due to an intrinsic defect in neutrophil phagocytosis or killing but as a result of reduced neutrophil numbers in the KO BAL. Interestingly, neutrophil numbers in the lung were increased in the KO compared with WT mice, suggesting a potential dysfunction in transepithelial migration of neutrophils from the lungs to the bronchoalveolar space. This effect was selective for lung tissue because peritoneal neutrophil numbers were similar between the two genotypes following peritoneal infection. Although neutrophil expression of CXCR2/CXCR3 was similar between WT and KO, IL-17A expression was higher in the KO compared with WT mice. These results suggest that enhanced neutrophil count in the KO lungs but reduced numbers in BAL are likely due to transepithelial migration defect and/or altered chemokines/cytokines. Together, our studies suggest a previously unrecognized and novel role for GRK6 in neutrophil migration specific to pulmonary tissue during bacterial infection.

Aims The arcuate nucleus is a vital brain region for coursing of pain command. G protein-coupled kinase 6 (GRK6) accommodates signaling through G protein-coupled receptors. Studies have demonstrated that GRK6 is involved in inflammatory pain and neuropathic pain. The present study was designed to explore the role and the underlying mechanism of GRK6 in arcuate nucleus of chronic visceral pain. Methods Chronic visceral pain of rats was induced by neonatal maternal deprivation and evaluated by monitoring the threshold of colorectal distension. Western blotting, immunofluorescence, real-time quantitative polymerase chain reaction techniques, and Nissl staining were employed to determine the expression and mutual effect of GRK6 with nuclear factor κB (NF-κB). Results Expression of GRK6 in arcuate nucleus was significantly reduced in neonatal maternal deprivation rats when compared with control rats. GRK6 was mainly expressed in arcuate nucleus neurons, but not in astrocytes, and a little in microglial cells. Neonatal maternal deprivation reduced the percentage of GRK6-positive neurons of arcuate nucleus. Overexpression of GRK6 by Lentiviral injection into arcuate nucleus reversed chronic visceral pain in neonatal maternal deprivation rats. Furthermore, the expression of NF-κB in arcuate nucleus was markedly upregulated in neonatal maternal deprivation rats. NF-κB selective inhibitor pyrrolidine dithiocarbamate suppressed chronic visceral pain in neonatal maternal deprivation rats. GRK6 and NF-κB were expressed in the arcuate nucleus neurons. Importantly, overexpression of GRK6 reversed NF-κB expression at the protein level. In contrast, injection of pyrrolidine dithiocarbamate once daily for seven consecutive days did not alter GRK6 expression in arcuate nucleus of neonatal maternal deprivation rats. Conclusions Present data suggest that GRK6 might be a pivotal molecule participated in the central mechanisms of chronic visceral pain, which might be mediated by inhibiting NF-κB signal pathway. Overexpression of GRK6 possibly represents a potential strategy for therapy of chronic visceral pain.

Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson's disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

Small molecules that inhibit the protein kinase A, G, and C (AGC) family of serine/threonine kinases can exert profound effects on cell homeostasis and thereby regulate fundamental processes such as heart rate, blood pressure, and metabolism, but there is not yet a clinically approved drug in the United States selective for a member of this family. One subfamily of AGC kinases, the G protein-coupled receptor (GPCR) kinases (GRKs), initiates the desensitization of active GPCRs. Of these, GRK2 has been directly implicated in the progression of heart failure. Thus, there is great interest in the identification of GRK2-specific chemical probes that can be further developed into therapeutics. Herein, we compare crystal structures of small molecule inhibitors in complex with GRK2 to those of highly selective compounds in complex with Rho-associated coiled-coil containing kinase 1 (ROCK1), a closely related AGC kinase. This analysis suggests that reduced hydrogen-bond formation with the hinge of the kinase domain, occupation of the hydrophobic subsite, and, consequently, higher buried surface area are key drivers of potency and selectivity among GRK inhibitors.

For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoidsin vivowas unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ9-THC), have delayed tolerance to Δ9-THC, and showed increased dependence for Δ9-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ9-THC was absent in S426A/S430A mutants. Δ9-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRsin vivo.

G protein-coupled receptor kinases (GRKs) are emerging as important integrative nodes in cell migration processes. Recent evidence links GRKs (particularly the GRK2 isoform) to the complex modulation of diverse aspects of cell motility. In addition to its well-established role in the desensitization of G protein-coupled receptors involved in chemotaxis, GRK2 can play a effector role in the organization of actin and microtubule networks and in adhesion dynamics, by means of novel substrates and transient interacting partners, such as the GIT-1 scaffold or the cytoplasmic α-tubulin deacetylase histone deacetylase 6 (HDAC6). The overall effect of altering GRK levels or activity on chemotaxis would depend on how such different roles are integrated in a given cell type and physiological context, and may have relevant implications in inflammatory diseases or cancer progression, Our laboratory is funded by grants from Ministerio de Educación y Ciencia (SAF2011-23800), Fundación Ramón Areces, The Cardiovascular Network (RECAVA) of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Comunidad de Madrid Indisnet Network (S2011/BMD-2332) to F.M, and Fundación Eugenio Rodriguez Pascual, Fundación Ramón Areces and Instituto de Salud Carlos III (PI11/00859) to P.P.

The interleukin-8 (IL-8/CXCL8) receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We have recently shown that CXCR1 couples predominantly to the G protein-coupled receptor (GPCR) kinase-2 (GRK2) whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to GPCRs, GRKs have displayed a more diverse protein/protein interaction in cells. In this study we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. The data herein demonstrated that upon CXCR2 activation, GRK6 interacts with activator of G protein signaling 3 (AGS3) and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time-dependent and peaked at 2-3 min post-activation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent but GRK6-independent fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca2+ mobilization, phosphoinositide (PI) hydrolysis and chemotaxis. In contrast, shRNA inhibition of AGS3 enhanced CXCL8-induced Ca2+ mobilization, receptor resistance to desensitization and recycling to the cell surface with no effect in receptor internalization. Interestingly, RBL-CXCR2-AGS3−/− cells displayed a significant increase in CXCR2 expression in the cell surface, but decreased (extracellular signal-regulated kinases) ERK1/2 and P38 mitogen-activated protein kinase (MAPK) activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels including downstream effector activation, receptor trafficking and expression at the cell membrane.

The classical model of G protein-coupled receptor (GPCR) activation is the two-state model, in which the GPCR exists in equilibrium between an active and inactive state. Based on this model, GPCR ligands have been classified as agonists, inverse agonists, or antagonists depending on their actions in shifting this equilibrium. Recently, however, accumulating evidence has indicated that GPCRs may exist in multiple active and inactive conformational states. In this situation, each ligand recognizes and stabilizes a specific conformation of the GPCR, leading to a set of specific biological effects. Based on this new model, a unique agonist or a combination of the usual agonist and an allosteric modulator may enable activation of a specific signaling pathway via a GPCR that activates multiple signals (biased agonism, functional selectivity). The calcium-sensing receptor autoantibody that we have identified in the serum of a patient with acquired hypocalciuric hypercalcemia (AHH) is the first example of a biased allosteric modulator of a GPCR working in a pathophysiological context. Our findings may indicate the presence of physiological allosteric modulators and provide new directions for the future drug development.

Background Recent studies have shown that expression of G protein-coupled receptor kinase 6 (GRK6) and β-arrestin1 in the striatum is closely associated with hyperactive dopamine receptors in rats with levodopa-induced dyskinesia (LID). Our research group has shown that Tianqi antitremor granules have a significant effect on the motor complications of Parkinson’s disease (PD). However, whether Tianqi antitremor granules have an effect on the behavioral manifestations and expression of GRK6 and β-arrestin1 in rats with LID is unknown. Methods Rats with PD received twice daily intraperitoneal injections of levodopa for 4 weeks to induce dyskinesia. Rats with LID were randomly divided into five groups: an LID-control group, an LID group, a levodopa plus Tianqi antitremor granules as traditional Chinese medicine (TCM)-low group, a levodopa plus TCM-medium group, and levodopa plus TCM-high group. Peak intensity of rotations was measured. GRK6 and β-arrestin1 expression in the striatum of the dyskinetic rats was observed by immunohistochemistry and Western blotting. Results Pulsatile treatment with levodopa induced abnormal involuntary movements in rats with PD similar to LID in patients with PD. We found that repeated levodopa administration increased peak rotations in dyskinetic rats. However, peak rotations were decreased in rats given levodopa plus the different doses of Tianqi antitremor granules. In accordance with changed behavior, GRK6 and β-arrestin1 expression was decreased in rats with PD and was persistently low in rats with LID, but this decrease was prevented by coadministration of levodopa and Tianqi antitremor granules. Conclusion Tianqi antitremor granules ameliorated levodopa-induced dyskinetic behavior, reversed the decrease in GRK6 and β-arrestin1 expression, and acted as a useful adjunctive medicine for the treatment of LID.

Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by G(s)-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the G(q) family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a G(q)-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a G(q)-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V(1b) vasopressin receptor, a G(q)-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V(1b) receptor antagonist led to reduced glucose excursions in a pyruvate challenge test. Taken together, these findings underscore the importance of G(q)-coupled receptors in regulating hepatic glucose fluxes and suggest novel receptor targets for the treatment of type 2 diabetes.

GRK-mediated Ser-129 phosphorylation of membrane-associated α-synuclein enhances dopamine uptake without altering cell surface expression of dopamine transporter. The data suggest a role of Ser-129–phosphorylated α-synuclein in modulating intracellular dopamine levels and damaging dopaminergic neurons in Parkinson disease., Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

In the present study, we investigated the mechanisms of brain derived neurotrophic factor (BDNF) in regulating cortical neuron premature synapse formation. KN-93, a specific inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), and G-protein-coupled receptor kinase interactor-1 (G1T1) siRNA were utilized, and the premature synapse formation of cortical neurons was detected under BDNF stimulation. Plasmids HA-GIT1, HA-GIT1 (ΔSLD), HA-GIT1 (S419A) and Flag-CaMKIIβ were constructed. The interaction between GIT1 and CaMKIIβ, and their influence on the premature synapse formation of BDNF-stimulated cortical neurons were examined. BDNF-stimulated cortical neurons were associated with increased premature synapse formation, the enhancement of phosphorylation for CaMKIIβ, and the combination of GIT1 and p-CaMKII(thr286). G1T1 siRNA and KN-93 inhibited premature synapse formation in cortical neurons. The interaction between GIT1 and CaMKIIβ required SLD domain and serine 419 in GIT1. BDNF-induced CaMKIIβ phosphorylation and premature synapse formation were suppressed in GIT1 (S419A) transfected cortical neurons. By interacting with CaMKIIβ, G1T1 (S419) were shown to participate in BDNF-induced premature synapse formation within cortical neurons.

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans. Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans, and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway.

Abnormal proliferation of endothelial cells (ECs) is important in vascular remodeling during hypertension, but the mechanisms are still unclear. In hypertensive rats caused by abdominal aortic coarctation, the expression of G-protein-coupled receptor kinase 6 (GRK6) in ECs at common carotid artery was repressed in vivo, and EC proliferation was increased. 15% cyclic stretch in vitro, which mimics the pathologically increased stretch in hypertension, repressed EC GRK6 expression via paracrine control by vascular smooth muscle cells (VSMCs). Furthermore, VSMC-derived microparticles (VSMC-MPs) were detected in the conditioned medium from VSMCs and in artery. VSMC-MPs from cells exposed to 15% cyclic stretch decreased GRK6 expression and increased EC proliferation. miR-27a was detected in VSMC-MPs and was upregulated by 15% cyclic stretch. miR-27a was transferred from VSMCs to ECs via VSMC-MPs and directly targeted on GRK6. Finally, a multi-point injection of antagomiR-27a around carotid artery decreased miR-27a expression in vivo, induced GRK6 expression, and reversed the abnormal EC proliferation. Pathologically elevated cyclic stretch increased the secretion of miR-27a, which was transferred from VSMCs to ECs via the VSMC-MPs, subsequently targeted GRK6, and induced EC proliferation. Locally decreasing miR-27a could be a novel therapeutic approach to attenuate the abnormal EC proliferation in hypertension.

Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein kinase A/G/C-like kinases, as relevant players in cancer progression, in a cell-type and tumor-specific way. Alterations in the expression and/or activity of particular GRKs have been identified in several types of tumors, and demonstrated to modulate the proliferation, survival or invasive properties of tumor cells by acting as integrating signaling nodes. GRKs are able to regulate the functionality of both G protein-coupled receptors (GPCR) and growth factor receptors and to directly control cytosolic, cytoskeletal or nuclear signaling components of pathways relevant for these processes. Furthermore, many chemokines as well as angiogenic and inflammatory factors present in the tumor microenvironment act through GPCR and other GRK-modulated signaling modules. Changes in the dosage of certain GRKs in the tumor stroma can alter tumor angiogenesis and the homing of immune cells, thus putting forward these kinases as potentially relevant modulators of the carcinoma-fibroblast-endothelial-immune cell network fostering tumor development and dissemination. A better understanding of the alterations in different GRK isoforms taking place during cancer development and metastasis in specific tumors and cell types and of its impact in signaling pathways would help to design novel therapeutic strategies.

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases that regulate a large and diverse class of G protein-coupled receptors (GPCRs). Through GRK phosphorylation and β-arrestin recruitment, GPCRs are desensitized and their signal terminated. Recent work on these kinases has expanded their role from canonical GPCR regulation to include noncanonical regulation of non-GPCR and nonreceptor substrates through phosphorylation as well as via scaffolding functions. Owing to these and other regulatory roles, GRKs have been shown to play a critical role in the outcome of a variety of physiological and pathophysiological processes including chemotaxis, signaling, migration, inflammatory gene expression, etc. This diverse set of functions for these proteins makes them popular targets for therapeutics. Role for these kinases in inflammation and inflammatory disease is an evolving area of research currently pursued in many laboratories. In this review, we describe the current state of knowledge on various GRKs pertaining to their role in inflammation and inflammatory diseases.

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels., Author summary Dopamine is a neurotransmitter that acts in the brain by binding seven transmembrane receptors that are coupled to heterotrimeric GTP-binding proteins (G proteins). Neuronal G proteins often function by modulating ion channels that control membrane excitability. Here we identify a molecular cascade downstream of dopamine in the nematode C. elegans that involves activation of the dopamine receptor DOP-3, activation of the G protein GOA-1, and inactivation of the NCA-1 and NCA-2 ion channels. We also identify a G protein-coupled receptor kinase (GRK-2) that inactivates the dopamine receptor DOP-3, thus leading to inactivation of GOA-1 and activation of the NCA channels. Thus, this study connects dopamine signaling to activity of the NCA channels through G protein signaling pathways.

To study the effect of G protein-coupled receptor kinase 6(GRK6) on proliferation and apoptosis of multiple myeloma(MM) cells and its mechanisms.The samples were collected from MM patients and healthy people for study in vivo. The plasma cells isolated from multiple myeloma patients, as well as U266 and NCI H929 myeloma cell lines were used for study in vitro. Western blot and quantitative real-time PCR were used to evaluate the protein and mRNA of expression of GRK6 in multiple myeloma, cell proliferation and apoptosis were tested by BrdU and Annexin V-FITC/PI assays.The protein and mRNA expression of GRK6 in multiple myeloma was higher than those in control group, and the expression level of GRK6 in stage I of MM was higher than that in control group, while the expression level of GRK6 in stage II was higher than that in stage I, but lower than that in stage III (P0.05). U266 and MM cells showed high-sensitivity to CX-4945, except NCI H929. GRK6 expression level in U266, NCI H929 and MM cells treated with siRNA and CX-4945, significantly decreased as compared with those cells treated by CX-4945 alone. Cell proliferations of U266, NCI H92 and MM groups treated with CX-4945 were (58.25±18.24)%, (64.32±20.03)% and (45.42±25.01)% respectively, moreover, their apoptotic rates were (62.82±53.21)%, (43.25±47.05)% and (85.67±40.32)% respectively.The expression level of GRK6 in multiple myeloma increases, and GRK6 inhibitor CX-4945 inhibits proliferation and promotes apoptosis of myeloma cells; GRK6 regulates Rac1 and involves in the proliferation and apoptosis pathway of multiple myeloma cells.

To explore the regulatory effect of GRK6 on the proliferation of multiple myeloma cells and its mechanism.A lentivirus vector shRNA interfering in human GRK6 gene expression was constructed and trans-fected into multiple myeloma cells to obtain the cell line MM1R with stable down-regulation of GRK6 gene expression. The real-time quantitative PCR and Western blot were used to confirm the effectiveness of the GRK6 gene expression down-regulation mediated by lentivirus vector. The MM1R cells with most obvious down-regulation were selected to detect the effect of GRK6 gene on cell proliferation.The lentivirus vector GRK6-shRNA interfering in human GRK6 gene was constructed succesufully and transfected into multiple myeloma cells, thereby the MM1R cell line with stable down-regulation of GRK6 gene was obtained. The CCK-8 assay showed that the proliferative viability of MM1R cells in experimental group was significantly lower than that in control group (P0.05); the flow cytometry showed that cells in experimental group were arrested in GA lentivirus vector which can specifically interfere in GRK6 gene expression is constructed successfully, The MM1R cell line with stable down-regulation of GRK6 expression is obtained by transfection and screening. The down-regulation of GRK6 expression can arrest MM1R cells in G

Summary Navigation of cells along gradients of guidance cues is a determining step in many developmental and immunological processes. Gradients can either be soluble or immobilized to tissues as demonstrated for the haptotactic migration of dendritic cells (DCs) toward higher concentrations of immobilized chemokine CCL21. To elucidate how gradient characteristics govern cellular response patterns, we here introduce an in vitro system allowing to track migratory responses of DCs to precisely controlled immobilized gradients of CCL21. We find that haptotactic sensing depends on the absolute CCL21 concentration and local steepness of the gradient, consistent with a scenario where DC directionality is governed by the signal-to-noise ratio of CCL21 binding to the receptor CCR7. We find that the conditions for optimal DC guidance are perfectly provided by the CCL21 gradients we measure in vivo. Furthermore, we find that CCR7 signal termination by the G-protein-coupled receptor kinase 6 (GRK6) is crucial for haptotactic but dispensable for chemotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. These findings suggest that stable, tissue-bound CCL21 gradients as sustainable "roads" ensure optimal guidance in vivo.

Background Pulmonary arterial hypertension (PAH) is defined as a complex disease of clinically characterized by elevated pulmonary pressure eventually resulting in right heart failure and premature death. To date, PAH still remains a life-threatening disease. Published evidence suggests that patients with PAH present profound sympathetic nervous system abnormalities and sympathetic activity has been shown to be increased. The mechanism of PAH is still complex and poorly understood. Results Some data have showed that adrenoceptors are involved in the process of the pathology and have different functions in the progression of PAH followed by heart failure. Alpha-adrenergic receptors mediate most excitatory effects and induce growth of smooth muscle cells and adventitial fibroblasts via complex cellular and molecular mechanisms. However, beta-adrenergic receptor mainly detected in endothelial layer commonly exerts relaxation effects on pulmonary artery. In addition, G protein-coupled receptor kinase 2, the primary G protein-coupled receptor kinase expressed in the heart, has been shown to be increased, resulting in the distinctive loss of inotropic reserve and functional capacity of the failing heart according to the activation of sympathetic nervous system. Conclusion Here, we summarize the relevant available studies describing the roles of sympathetic nervous system in the progression of PAH.

Multiple organ systems require epithelial barriers for normal function, and barrier loss is a hallmark of diseases ranging from inflammation to epithelial cancers. However, the molecular processes regulating epithelial barrier maturation are not fully elucidated. After contact, epithelial cells undergo size-reductive proliferation and differentiate, creating a dense, highly ordered monolayer with high resistance barriers. We provide evidence that the tight junction protein occludin contributes to the regulation of epithelial cell maturation upon phosphorylation of S471 in its coiled-coil domain. Overexpression of a phosphoinhibitory occludin S471A mutant prevents size-reductive proliferation and subsequent tight junction maturation in a dominant manner. Inhibition of cell proliferation in cell-contacted but immature monolayers recapitulated this phenotype. A kinase screen identified G-protein-coupled receptor kinases (GRKs) targeting S471, and GRK inhibitors delayed epithelial packing and junction maturation. We conclude that occludin contributes to the regulation of size-reductive proliferation and epithelial cell maturation in a phosphorylation-dependent manner.

It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes tumor growth and angiogenesis. In this study, we demonstrate that c-Src, IGF-IR and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (SrcpY416) is co-expressed in Exo with phosphorylated FAK (FAKpY861), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, SrcpY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo.

G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes.

Hypertension, a complex trait determined by genetic, epigenetic, and environmental factors and their intricate interaction, is an important public health challenge worldwide because of its high prevalence and concomitant increase in the risk for cardiovascular disease. Unfortunately, the prevalence

C-kit positive cardiac stem cells (CSCs) have been shown to contribute to myocardial regeneration after infarction. Previously, we have shown that the c-kit ligand stem cell factor (SCF) can induce CSC migration into the infarcted area during myocardial infarction (MI). However, the precise mechanism involved is not fully understood. In this study, we found that CSCs also express C-X-C chemokine receptor type 4 (CXCR4), which is a typical member of the seven transmembrane-spanning G protein-coupled receptor (GPCR). In vitro, activation of c-kit signalling by SCF promotes migration of CSCs with increased phosphorylation of CXCR4-serine 339, p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinases 1/2 (ERK1/2). Knockdown of CXCR4 expression by siRNA reduces SCF/c-kit-induced migration and downstream signalling. As previously reported, CXCR4-serine 339 phosphorylation is mainly regulated by GPCR kinase 6 (GRK6); thus, silencing of GRK6 expression by siRNA impairs CXCR4-serine 339 phosphorylation and migration of CSCs caused by SCF. In vivo, knockdown of GRK6 impairs the ability of CSCs to migrate into peri-infarcted areas. These results demonstrate that SCF-induced CSC migration is regulated by the transactivation of CXCR4-serine 339 phosphorylation, which is mediated by GRK6.

Actions of the neurotransmitter dopamine in the brain are mediated by dopamine receptors that belong to the superfamily of G protein-coupled receptors (GPCRs). Mammals have five dopamine receptor subtypes, D1 through D5. D1 and D5 couple to Gs/olf and activate adenylyl cyclase, whereas D2, D3, and D4 couple to Gi/o and inhibit it. Most GPCRs upon activation by an agonist are phosphorylated by GPCR kinases (GRKs). The GRK phosphorylation makes receptors high-affinity binding partners for arrestin proteins. Arrestin binding to active phosphorylated receptors stops further G protein activation and promotes receptor internalization, recycling or degradation, thereby regulating their signaling and trafficking. Four non- visual GRKs are expressed in striatal neurons. Here we describe known effects of individual GRKs on dopamine receptors in cell culture and in the two in vivo models of dopamine-mediated signaling: behavioral response to psychostimulants and L-DOPA- induced dyskinesia. Dyskinesia, associated with dopamine super-sensitivity of striatal neurons, is a debilitating side effect of L-DOPA therapy in Parkinson's disease. In vivo, GRK subtypes show greater receptor specificity than in vitro or in cultured cells. Overexpression, knockdown, and knockout of individual GRKs, particularly GRK2 and GRK6, have differential effects on signaling of dopamine receptor subtypes in the brain. Furthermore, deletion of GRK isoforms in select striatal neuronal types differentially affects psychostimulant-induced behaviors. In addition, anti-dyskinetic effect of GRK3 does not require its kinase activity: it is mediated by the binding of its RGS-like domain to Gαq/11, which suppresses Gq/11 signaling. The data demonstrate that the dopamine signaling in defined neuronal types in vivo is regulated by specific and finely orchestrated actions of GRK isoforms.

Activation of a wheat gene product by a fungal protein leads to cell death in the plant, allowing the pathogen to cause disease., Necrotrophic pathogens live and feed on dying tissue, but their interactions with plants are not well understood compared to biotrophic pathogens. The wheat Snn1 gene confers susceptibility to strains of the necrotrophic pathogen Parastagonospora nodorum that produce the SnTox1 protein. We report the positional cloning of Snn1, a member of the wall-associated kinase class of receptors, which are known to drive pathways for biotrophic pathogen resistance. Recognition of SnTox1 by Snn1 activates programmed cell death, which allows this necrotroph to gain nutrients and sporulate. These results demonstrate that necrotrophic pathogens such as P. nodorum hijack host molecular pathways that are typically involved in resistance to biotrophic pathogens, revealing the complex nature of susceptibility and resistance in necrotrophic and biotrophic pathogen interactions with plants.

Berberine (BBR), an effective compound of Chinese traditional herbal medicine, has preventive effects on diabetes and its complications. In this study, we investigated the therapeutic effects and underlying molecular mechanisms of BBR in rats with high-fat diet and streptozotocin (STZ)-induced diabetic nephropathy model. BBR (50, 100, 200 mg/kg/d) were orally administered to male Sprague-Dawley rats after STZ injection and conducted for 8 weeks. Renal damage was evaluated by kidney weight to body weight ratio (KW/BW), urine microalbumin (UMAlb), urine protein for 24 h (UP24 h), urine creatinine (UCr), and histological examination. Type IV collagen and transforming growth factor-beta1 (TGF-β1) were detected by immunohistochemistry and ultrastructure of glomeruli was observed. Fasting blood glucose (FBG),serum creatinine (SCr), blood urea nitrogen (BUN), total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-c), low-density lipoprotein-cholesterol (LDL-c) in serum and G protein-coupled receptor kinases (GRKs), cAMP in kidney were measured. Remarkable renal damage, hyperglycemia and hyperlipidemia were observed in DN rats. BBR could restore renal functional parameters, suppress alterations in histological and ultrastructural changes in the kidney tissues, improve glucose and lipid metabolism disorders, and increase cAMP levels compared with those of DN model group. Furthermore, BBR down-regulated total protein expression of GRK2, GRK3 and up-regulated expression of GRK6 of renal cortex in DN rats, but had a slight effects on GRK4 and GRK5. These studies demonstrate, for the first time, that BBR exerts renoprotection in high-fat diet and STZ-induced DN rats by modulating the proteins expression of GRKs in G protein- AC-cAMP signaling pathway.

Circadian clocks provide a temporal structure to processes from gene expression to behavior in organisms from all phyla. Most clocks are synchronized to the environment by alternations of light and dark. However, many organisms experience only muted daily environmental cycles due to their lightless spatial niches (e.g., caves or soil). This has led to speculation that they may dispense with the daily clock. However, recent reports contradict this notion, showing various behavioral and molecular rhythms in Caenorhabditis elegans and in blind cave fish. Based on the ecology of nematodes, we applied low-amplitude temperature cycles to synchronize populations of animals through development. This entrainment regime reveals rhythms on multiple levels: in olfactory cued behavior, in RNA and protein abundance, and in the oxidation state of a broadly conserved peroxiredoxin protein. Our work links the nematode clock with that of other clock model systems; it also emphasizes the importance of daily rhythms in sensory functions that are likely to impact on organism fitness and population structure.

OBJECTIVE To investigate the effects of Tianqi Pingchan (TQPC) Granule, a compound traditional Chinese herbal medicine with antitremor activity, on levodopa-induced dyskinesia and the expression of G protein-coupled receptor kinase 6 (GRK6) in rats with Parkinson disease (PD). METHODS The hemi-Parkinsonian rat model was established by sterotaxically injecting 6-hydroxydopa (6-OHDA) to the right medial forebrain bundle. Rats with PD were randomly divided into 5 groups with 5 in each. PD group was intraperitoneally injected with vitamin C; levodopa group was intraperitoneally injected with levodopa and benserazide; low-, medium- and high-dose TQPC Granule groups were intraperitoneally injected with levodopa and benserazide and treated with different dosages of TQPC Granule by gavage for 29 d. Another 5 rats were served as control with sham-operation. The behaviors of rats were observed and classified with abnormal involuntary movement (AIM) score. The expression of GRK6 in the striate of rats was detected by immunohistochemical method and Western blotting. RESULTS AIM score was increased and the expression of GRK6 protein in lesion side was decreased after the long-tern treatment with levodopa and benserazide in rats. The AIM scores of rats with PD were decreased after TGPC Granule treatment. Immunohistochemical results showed that the number of GRK6-positive cells in medium- and high-dose TQPC Granule groups was increased as compared to that in the levodopa group (P

Hedgehog (Hh) signaling is essential for proper tissue patterning and maintenance and has a substantial impact on human disease. While many of the main components and mechanisms involved in transduction of the Hh signal have been identified, the details of how the pathway functions are continually being refined. One aspect that has attracted much attention recently is the involvement of G-protein-coupled receptor kinases (GRKs) in the pathway. These regulators of G-protein-coupled receptor (GPCR) signaling have an evolutionarily-conserved function in promoting high-threshold Hh target gene expression through regulation of Smoothened (Smo), a GPCR family member that activates intracellular Hh signaling. Several models of how GRKs impact on Smo to increase downstream signaling have been proposed. Recently, we demonstrated that these kinases have surprisingly complex and conflicting roles, acting to limit signaling through the pathway while also promoting Smo activity. In addition to the previously described direct effects of Gprk2 on Smo activation, Gprk2 also indirectly affects Hh signaling by controlling production of the second messenger cyclic AMP to influence Protein kinase A activity.

Background Cannabinoid CB 1 receptors (CB 1 Rs) mediate the effects of ▵ 9 -tetrahydrocannabinol (THC), the psychoactive component in marijuana. Repeated THC administration produces tolerance and dependence, which limit therapeutic development. Moreover, THC produces motor and psychoactive side effects. β-arrestin2 mediates receptor desensitization, internalization, and signaling, but its role in these CB 1 R effects and receptor regulation is unclear. Methods CB 1 R signaling and behaviors (antinociception, hypothermia, catalepsy) were assessed in β-arrestin2-knockout (βarr2-KO) and wild-type mice after THC administration. Cannabinoid-stimulated [ 35 S]GTPγS and [ 3 H]ligand autoradiography were assessed by statistical parametric mapping and region-of-interest analysis. Results β-arrestin2 deletion increased CB 1 R-mediated G-protein activity in subregions of the cortex but did not affect CB 1 R binding, in vehicle-treated mice. βarr2-KO mice exhibited enhanced acute THC-mediated antinociception and hypothermia, with no difference in catalepsy. After repeated THC administration, βarr2-KO mice showed reduced CB 1 R desensitization and/or downregulation in cerebellum, caudal periaqueductal gray, and spinal cord and attenuated tolerance to THC-mediated antinociception. In contrast, greater desensitization was found in hypothalamus, cortex, globus pallidus, and substantia nigra of βarr2-KO compared with wild-type mice. Enhanced tolerance to THC-induced catalepsy was observed in βarr2-KO mice. Conclusions β-arrestin2 regulation of CB 1 R signaling following acute and repeated THC administration was region-specific, and results suggest that multiple, overlapping mechanisms regulate CB 1 Rs. The observations that βarr2-KO mice display enhanced antinociceptive responses to acute THC and decreased tolerance to the antinociceptive effects of the drug, yet enhanced tolerance to catalepsy, suggest that development of cannabinoid drugs that minimize CB 1 R interactions with β-arrestin2 might produce improved cannabinoid analgesics with reduced motor suppression.

Heptahelical G-protein-coupled receptors are the most diverse and therapeutically important family of receptors, playing major roles in the physiology of various organs and tissues. They couple their ligand binding to G-protein activation, which then transmits intracellular signals. G-protein signaling is terminated by phosphorylation of the receptor by the family of G-protein-coupled receptor kinases (GRKs), followed by arrestin (Arr) binding, which uncouples the phosphorylated receptor from the G-protein and subsequently targets the receptor for internalization. Moreover, Arrs can transmit signals in their own right during receptor internalization. Genetic polymorphisms in receptors, as well as in GRK and Arr family members per se, which affect regulation of receptor signaling and function, have just started being identified and characterized. The present review will discuss what is known so far in this evolving field of GRK/Arr pharmacogenomics, as well as highlight important areas likely to produce invaluable information in the future.

We examined the regulatory mechanisms of the affinity of Gq protein-coupled histamine H1-receptors for histamine after histamine pretreatment in intact human U373 MG astrocytoma cells. In control cells, the displacement curves for histamine against the binding of 5 nM [3H] mepyramine, a radioligand for H1-receptors, showed the presence of two binding sites for histamine, that is, high and low affinity sites. Pretreatment with 0.1 mM histamine for 30 min at 37°C induced a significant reduction in the percentage of high affinity sites for histamine and a concomitant increase in the percentage of low affinity sites with no change in their pIC50 values. These histamine-induced changes were insensitive to 30 μM KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II, but they were completely inhibited either by 0.4 mM ZnCl2, an inhibitor of G protein-coupled receptor kinases (GRKs), or under hypertonic conditions, where clathrin-mediated endocytosis is known to be inhibited. These results suggest that histamine-induced conversion of high to low affinity sites for histamine is predominantly regulated by GRK/clathrin-mediated internalization of H1-receptors in human astrocytoma cells. Keywords:: agonist affinity, desensitization, G protein-coupled receptor, histamine H1-receptor, internalization

Heptahelical, G protein-coupled or seven transmembrane-spanning receptors, such as the β-adrenergic and the angiotensin II type 1 receptors, are the most diverse and therapeutically important family of receptors in the human genome, playing major roles in the physiology of various organs/tissues including the heart and blood vessels. Ligand binding activates heterotrimeric G proteins that transmit intracellular signals by regulating effector enzymes or ion channels. G protein signaling is terminated, in large part, by phosphorylation of the agonist-bound receptor by the G-protein coupled receptor kinases (GRKs), followed by βarrestin binding, which uncouples the phosphorylated receptor from the G protein and subsequently targets the receptor for internalization. As the receptor-βarrestin complex enters the cell, βarrestin-1 and -2, the two mammalian βarrestin isoforms, serve as ligand-regulated scaffolds that recruit a host of intracellular proteins and signal transducers, thus promoting their own wave of signal transduction independently of G-proteins. A constantly increasing number of studies over the past several years have begun to uncover specific roles played by these ubiquitously expressed receptor adapter proteins in signal transduction of several important heptahelical receptors regulating the physiology of various organs/ systems, including the cardiovascular (CV) system. Thus, βarrestin-dependent signaling has increasingly been implicated in CV physiology and pathology, presenting several exciting opportunities for therapeutic intervention in the treatment of CV disorders. Additionally, the discovery of this novel mode of heptahelical receptor signaling via βarrestins has prompted a revision of classical pharmacological concepts such as receptor agonism/antagonism, as well as introduction of new terms such as "biased signaling", which refers to ligand-specific activation of selective signal transduction pathways by the very same receptor. The present review gives an overview of the current knowledge in the field of βarrestin-dependent signaling, with a specific focus on CV heptahelical receptor βarrestin-mediated signaling and on "biased" CV heptahelical receptor ligands that promote or inhibit it. Exciting new possibilities for cardiovascular therapeutics arising from the delineation of this βarrestin-dependent signaling are also discussed.

In heart failure (HF), sympathetic hyperactivation induces deleterious effects in myocardial β-adrenergic signaling, with receptor down-regulation and desensitization mediated by G protein receptor-coupled kinases (GRKs). We hypothesised that changes in GRK isoforms may be associated with clinical status in advanced HF, using the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) scale.We included 31 patients with advanced HF undergoing transplantation. According to INTERMACS profiles, mRNA and protein levels of GRK isoforms in left ventricular (LV) myocardium were analyzed and compared with nonfailing LV samples.In failing LV myocardium, GRK2 and GRK5 (but not GRK3) protein was up-regulated compared with control samples. Among HF patients, an increase in GRK2 and GRK5 mRNA and protein abundance was observed in β-agonist-treated patients (vs β-blockers: P.05) and in higher-risk INTERMACS status (profiles 2 and 3 vs 4 and 5: P.05). A significant negative correlation of GRK2 expression with LV stroke volume supported these findings.Increased GRK2 correlates with clinical severity using the INTERMACS scale and LV stroke volume, supporting it as a potential target in advanced HF. These changes are paralleled by GRK5 expression in the failing myocardium, suggesting a relevant role in human HF.