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During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G‐protein‐coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation‐related genes through nuclear factor‐κB activation, leading to an increase in the expression levels of fibrosis‐related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5‐mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared with that in wild‐type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI.

Abstract Background G-protein-coupled receptor (GPCR) kinases (GRKs) interact with ligand-activated GPCR, causing intracellular phosphorylation and interfering with the intracellular signal transduction associated with the development of cancer. Colorectal cancer (CRC) is a fast-growing disease, and its molecular mechanism involves various regulatory proteins, including kinases. However, the GRK mechanism in CRC has not been explored. Methods We used an integrated computational approach to investigate the potential of GRK family members as targeted proteins in CRC. The GRK expression levels in tumor and normal tissues, colon adenocarcinoma samples, and metastatic colon adenocarcinoma were analyzed using ONCOMINE, GEPIA, and UALCAN, as well as TNM plots. Genetic changes in the GRK family genes were investigated using cBioportal. The prognostic value related to the gene expression of the GRK family was examined using GEPIA and UALCAN. Co-expression analysis of the GRK family was conducted using COXPRESdb. Association analysis of the Gene Ontology, KEGG pathway enrichment, and drug-gene analyses were performed using the over-representation analysis (ORA) in WebGestalt. Results GRK2, GRK3, and GRK5 mRNA levels increased significantly in patients with CRC and metastatic CRC. Genetic changes were detected in patients with CRC, including GRK7 (1.1%), GRK2 (1.7%), GRK4 (2.3%), GRK5 (2.5%), GRK6 (2.5%), GRK3 (2.9%), and GRK1 (4%). CRC patients with low mRNA of GRK7 levels had better disease-free and overall survival than those with high GRK7 levels. Hierarchical clustering analysis revealed significant positive correlations between GRK5 and GRK2 and between GRK2 and GRK6. KEGG pathway enrichment analysis showed that the gene network (GN) regulated several cellular pathways, such as the morphine addiction signaling and chemokine signaling pathways in cancer. The drug-gene association analysis indicated that the GN was associated with several drugs, including reboxetine, pindolol, beta-blocking agents, and protein kinase inhibitors. Conclusion No research has been conducted on the relation of GRK1 and GRK7 to cancer, particularly CRC. In this work, genes GRK2, GRK3, GRK5, and GRK6 were found to be oncogenes in CRC. Although inhibitors against GRK2, GRK5, and GRK6 have previously been developed, further research, particularly preclinical and clinical studies, is needed before these agents may be used to treat CRC.

During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G‐protein‐coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation‐related genes through nuclear factor‐κB activation, leading to an increase in the expression levels of fibrosis‐related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5‐mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared with that in wild‐type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI. [ABSTRACT FROM AUTHOR]

Abstract In the pineal organ of zebrafish larvae, the bistable opsin parapinopsin alone generates color opponency between UV and visible light. Our previous study suggested that dark inactivation of the parapinopsin photoproduct, which activates G-proteins, is important for the regulation of the amount of the photoproduct. In turn, the photoproduct is responsible for visible light sensitivity in color opponency. Here, we found that an opsin kinase or a G-protein-coupled receptor kinase (GRK) is involved in inactivation of the active photoproduct of parapinopsin in the pineal photoreceptor cells of zebrafish larvae. We investigated inactivation of the photoproduct in the parapinopsin cells of various knockdown larvae by measuring the light responses of the cells using calcium imaging. We found that GRK7a knockdown slowed recovery of the response of parapinopsin photoreceptor cells, whereas GRK1b knockdown or GRK7b knockdown did not have a remarkable effect, suggesting that GRK7a, a cone-type GRK, is mainly responsible for inactivation of the parapinopsin photoproduct in zebrafish larvae. We also observed a similar knockdown effect on the response of the parapinopsin photoreceptor cells of mutant larvae expressing the opsin SWS1, a UV-sensitive cone opsin, instead of parapinopsin, suggesting that the parapinopsin photoproduct was inactivated in a way similar to that described for cone opsins. We confirmed the immunohistochemical distribution of GRK7a in parapinopsin photoreceptor cells by comparing the immunoreactivity to GRK7 in GRK7a-knockdown and control larvae. These findings suggest that in pineal photoreceptor cells, the cone opsin kinase GRK7a contributes greatly to the inactivation of parapinopsin, which underlies pineal color opponency.

In the pineal organ of zebrafish larvae, the bistable opsin parapinopsin alone generates color opponency between UV and visible light. Our previous study suggested that dark inactivation of the parapinopsin photoproduct, which activates G-proteins, is important for the regulation of the amount of the photoproduct. In turn, the photoproduct is responsible for visible light sensitivity in color opponency. Here, we found that an opsin kinase or a G-protein-coupled receptor kinase (GRK) is involved in inactivation of the active photoproduct of parapinopsin in the pineal photoreceptor cells of zebrafish larvae. We investigated inactivation of the photoproduct in the parapinopsin cells of various knockdown larvae by measuring the light responses of the cells using calcium imaging. We found that GRK7a knockdown slowed recovery of the response of parapinopsin photoreceptor cells, whereas GRK1b knockdown or GRK7b knockdown did not have a remarkable effect, suggesting that GRK7a, a cone-type GRK, is mainly responsible for inactivation of the parapinopsin photoproduct in zebrafish larvae. We also observed a similar knockdown effect on the response of the parapinopsin photoreceptor cells of mutant larvae expressing the opsin SWS1, a UV-sensitive cone opsin, instead of parapinopsin, suggesting that the parapinopsin photoproduct was inactivated in a way similar to that described for cone opsins. We confirmed the immunohistochemical distribution of GRK7a in parapinopsin photoreceptor cells by comparing the immunoreactivity to GRK7 in GRK7a-knockdown and control larvae. These findings suggest that in pineal photoreceptor cells, the cone opsin kinase GRK7a contributes greatly to the inactivation of parapinopsin, which underlies pineal color opponency. [ABSTRACT FROM AUTHOR]

G-protein βγ subunits are key participants in G-protein signaling. These subunits facilitate interactions between receptors and G proteins that are critical for the G protein activation cycle at the plasma membrane. In addition, they play roles in directly transducing signals to an ever expanding range of downstream targets, including integral membrane and cytosolic proteins. Emerging data indicate that Gβγ may play additional roles at intracellular compartments including endosomes, the Golgi apparatus, and the nucleus. Here, we discuss the molecular and structural basis for their ability to coordinate this wide range of cellular activities. [ABSTRACT FROM AUTHOR]

Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity.

Objectives: Molecular mechanisms linking autonomic dysfunction with poorer clinical outcomes in critical illness remain unclear. We hypothesized that baroreflex dysfunction alone is sufficient to cause cardiac impairment through neurohormonal activation of (nicotinamide adenine dinucleotide phosphate oxidase dependent) oxidative stress resulting in increased expression of G-protein-coupled receptor kinase 2, a key negative regulator of cardiac function.Design: Laboratory/clinical investigations.Setting: University laboratory/medical centers.Subjects: Adult rats; wild-type/nicotinamide adenine dinucleotide phosphate oxidase subunit-2-deficient mice; elective surgical patients.Interventions: Cardiac performance was assessed by transthoracic echocardiography following experimental baroreflex dysfunction (sino-aortic denervation) in rats and mice. Immunoblots assessed G-protein-coupled receptor recycling proteins expression in rodent cardiomyocytes and patient mononuclear leukocytes. In surgical patients, heart rate recovery after cardiopulmonary exercise testing, time/frequency measures of parasympathetic variables were related to the presence/absence of baroreflex dysfunction (defined by spontaneous baroreflex sensitivity of <6 ms mm Hg). The associations of baroreflex dysfunction with intraoperative cardiac function and outcomes were assessed.Measurements and Main Results: Experimental baroreflex dysfunction in rats and mice resulted in impaired cardiac contractility and upregulation of G-protein-coupled receptor kinase 2 expression. In mice, genetic deficiency of gp91 nicotinamide adenine dinucleotide phosphate oxidase subunit-2 prevented upregulation of G-protein-coupled receptor kinase 2 expression in conditions of baroreflex dysfunction and preserved cardiac function. Baroreflex dysfunction was present in 81 of 249 patients (32.5%) and was characterized by lower parasympathetic tone and increased G-protein-coupled receptor kinase 2 expression in mononuclear leukocytes. Baroreflex dysfunction in patients was also associated with impaired intraoperative cardiac contractility. Critical illness and mortality were more frequent in surgical patients with baroreflex dysfunction (relative risk, 1.66 [95% CI, 1.16-2.39]; p = 0.006).Conclusions: Reduced baroreflex sensitivity is associated with nicotinamide adenine dinucleotide phosphate oxidase subunit-2-mediated upregulation of G-protein-coupled receptor kinase 2 expression in cardiomyocytes and impaired cardiac contractility. Autonomic dysfunction predisposes patients to the development of critical illness and increases mortality. [ABSTRACT FROM AUTHOR]

The signaling of seven transmembrane receptors/G-protein- coupled receptors (GPCRs) is regulated by a number of receptor interacting proteins, including βarrestins (βarrs) and GPCR kinases (GRKs). In the present report, we have analyzed the interaction pattern between the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), βarr2, and GRK2 using bioluminescence resonance energy transfer assays. We found that βarr2 interacts with the GLP-1R in a biphasic manner with a phosphorylation-independent and a phosphorylation-dependent component. In competition experiments, we observed βarr2 competing with GRK2 for interaction with GLP-1R. We propose a model were βarr2 competes with GRK2 for interaction with the activated and GRK phosphorylated GLP-1R, suggesting a new role of βarr2 in regulating the orchestration of GRK2 functionality. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The ubiquitin-proteasome system (UPS) plays a central role in protein degradation and regulates a variety of critical cellular processes. During recent years, the cardiac UPS has become increasingly recognized as a key regulator of cardiac function under both physiological and pathological conditions. Numerous studies have demonstrated that altered UPS function is involved in the pathogenesis of cardiac disease including myocardial ischaemia or infarction. The expression and activity of the E3 ubiquitin ligases, which confer substrate specificity in the UPS pathway, affect the apoptosis and severity of disease in myocardial ischaemia and reperfusion. Although impaired proteasome function is commonly associated with myocardial ischaemic injury, recent evidence also supports a cardioprotective role for proteasome inhibitors in myocardial ischaemia. We will review these studies and data, discuss controversies regarding the UPS alterations and use of proteasome inhibitors in myocardial ischaemia, and attempt to identify strategies that may enhance their clinical application. [ABSTRACT FROM PUBLISHER]

Abstract: The Hedgehog (Hh) signaling pathway plays a conserved and essential role in regulating development and homeostasis of numerous tissues. Cytoplasmic signaling is initiated by Smoothened (Smo), a G-protein-coupled receptor (GPCR) family member, whose levels and activity are regulated by the Hh receptor Patched (Ptc). In response to Hh binding to Ptc, Ptc-mediated repression of Smo is relieved, leading to Smo activation, surface accumulation, and downstream signaling. We find that downregulation of Drosophila Smo protein in Hh-responding imaginal disc cells is dependent on the activity of G-protein-coupled receptor kinase 2 (Gprk2). By analyzing gain- and null loss-of-function phenotypes, we provide evidence that Gprk2 promotes Smo internalization subsequent to its activation, most likely by direct phosphorylation. Ptc-dependent regulation of Smo accumulation is normal in gprk2 mutants, indicating that Gprk2 and Ptc downregulate Smo by different mechanisms. Finally, we show that both Drosophila G-protein-coupled receptor kinase orthologues, Gprk1 and Gprk2, act in a partially redundant manner to promote Hh signaling. Our results suggest that Smo is regulated by distinct Ptc-dependent and Gprk2-dependent trafficking mechanisms in vivo, analogous to constitutive and activity-dependent regulation of GPCRs. G-protein-coupled receptor kinase activity is also important for efficient downstream signaling. [Copyright &y& Elsevier]

Abstract: The majority of α-synuclein (αS) deposited in Lewy bodies, the pathological hallmark of Parkinson’s disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of αS has been demonstrated to enhance the αS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of αS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of αS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of αS; however, knockdown of GRK2 or GRK5 did not decrease αS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of αS in HEK293 cells. [Copyright &y& Elsevier]

Many proteins are modified by the covalent addition of different types of lipids, such as myristoylation, palmitoylation and prenylation. Lipidation is expected to promote membrane association of proteins. Visual phototransduction involves many lipid-modified proteins. The G-Protein-coupled receptor of rod photoreceptors, rhodopsin, is inactivated by G-Protein-coupled Receptor Kinase 1 (GRK1). The C-terminus of GRK1 is farnesylated and its truncation has been shown to result in a very high decrease of its enzymatic activity, most likely because of the loss of its membrane localization. Little information is available on the membrane binding of GRK1 as well as of most prenylated proteins. Measurements of the membrane binding of the non-farnesylated and farnesylated C-terminal segment of GRK1 were thus performed using lipids typical of those found in rod outer segment disk membranes. Their random coil secondary structure was determined using circular dichroism and infrared spectroscopy. The non-farnesylated C-terminal segment of GRK1 has no surface activity. In contrast, the farnesylated C-terminal segment of GRK1 shows a particularly strong binding to lipid monolayers bearing at least one unsaturated fatty acyl chain. No binding is observed in the presence of monolayers of saturated phospholipids, in agreement with the low affinity of farnesylated Ras proteins for lipids in the liquid-ordered state. Altogether, these data demonstrate that the farnesyl group of the C-terminal segment of GRK1 is mandatory for its membrane binding, which is favored by particular lipids or lipid mixtures. This information will also be useful for the understanding of the membrane binding of other prenylated proteins. [Display omitted] • The C-terminal segment of the protein GRK1 (Cter-GRK1) has a random coil structure. • Non-Farnesylated Cter-GRK1 has no surface activity. • Farnesylated Cter-GRK1 does not bind monolayers of saturated phospholipids and DRM. • High membrane binding of Farnesylated Cter-GRK1 to unsaturated lipid monolayers. [ABSTRACT FROM AUTHOR]

Almost all G-protein coupled receptors (GPCRs) are regulated by phosphorylation and this process is a key event in determining the signalling properties of this receptor super-family. Receptors are multiply phosphorylated at sites that can occur throughout the intracellular regions of the receptor. This diversity of phospho-acceptor sites together with a lack of consensus phosphorylation sequences has led to the suggestion that the precise site of phosphorylation is not important in the phosphorylation-dependent regulation of GPCR function but rather it is the increase in bulk negative charge of the intracellular face of the receptor which is the significant factor. This review investigates the possibility that the multi-site nature of GPCR phosphorylation reflects the importance of specific phosphorylation events which mediate distinct signalling outcomes. In this way receptor phosphorylation may provide for a flexible regulatory mechanism that can be tailored in a tissue specific manner to regulate physiological processes. By understanding the flexible nature of GPCR phosphorylation if may be possible to develop agonists or allosteric modulators that promote a subset of phosphorylation events on the target GPCR and thereby restrict the action of the drug to a particular receptor mediated signalling response. [ABSTRACT FROM AUTHOR]

The molecular mechanisms mediating stress-induced dysphoria in humans and conditioned place aversion in rodents are unknown. Here, we show that repeated swim stress caused activation of both κ-opioid receptor (KOR) and p38 mitogen-activated protein kinase (MAPK) coexpressed in GABAergic neurons in the nucleus accumbens, cortex, and hippocampus. Sites of activation were visualized using phosphoselective antibodies against activated κ receptors (KOR-P) and against phospho-p38 MAPK. Surprisingly, the increase in P-p38-IR caused by swim-stress exposure was completely KOR dependent; P-p38-IR did not increase in KOR(-/-) knock-out mice subjected to the same swim-paradigm or in wild-type mice pretreated with the KOR antagonist norbinaltorphimine. To understand the relationship between p38 activation and the behavioral effects after KOR activation, we administered the p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (i.c.v.)] and found that it selectively blocked the conditioned place aversion caused by the κ agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and the KOR-dependent swim stress-induced immobility while not affecting κ-opioid analgesia or nonselectively affecting associative learning. We found that the mechanism linking KOR and p38 activation in vivo was consistent with our previous in vitro data suggesting that β-arrestin recruitment is required; mice lacking G-protein-coupled receptor kinase 3 also failed to increase p-p38-IR after KOR activation in vivo, failed to show swim stress-induced immobility, or develop conditioned place aversion to U50488. Our results indicate that activation of p38 MAPK signaling by the endogenous dynorphin-κ-opioid system likely constitutes a key component of the molecular mechanisms mediating the aversive properties of stress. [ABSTRACT FROM AUTHOR]

Partial sciatic nerve ligation (pSNL) markedly increased glial fibrillary acidic protein immunoreactivity (GFAP-IR) 1 week after lesion in the L4-L5 spinal dorsal horn of wild-type, but not in dynorphin knock-out, mice lacking κ opioid receptors (KOR-/-) or in wild-type mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). A direct effect of KOR on glial cell proliferation was suggested by the findings that primary cultures of type II GFAP-immunoreactive astrocytes isolated from mouse spinal cord express KOR. Sustained treatment with the κ agonist U50,488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate) significantly increased the proliferation rate of GFAP-immunoreactive astrocytes isolated from wild-type mice, and this effect was blocked by norBNI pretreatment. Proliferation of cultured type II astrocytes may have been stimulated by mitogen-activated protein kinase (MAPK) activation by KOR because (1) U50,488 treatment increased phospho-p38 MAPK-immunoreactivity 247 ± 44% over untreated cells, (2) the increase in phospho-p38 induced by U50,488 was blocked by norBNI and not evident in KOR-/- cultures, and (3) GFAP-immunoreactive astrocyte proliferation induced by U50,488 was blocked by the p38 MAPK inhibitor SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Similar mechanisms of astrocyte activation may also be responsible in vivo because intrathecal injection of SB 203580 blocked the increased GFAP-IR in lumbar spinal cord induced by pSNL. Although the relationship between κ-stimulated astrocyte proliferation and neuropathic pain mechanisms was not directly established in these studies, the results support the hypothesis that KOR activation induces spinal astrocyte proliferation, which may contribute to cellular reorganization after sciatic nerve damage. [ABSTRACT FROM AUTHOR]

There is a significant gap between advances in medication for mental disorders and the present static situation of biological diagnosis and monitoring treatment. The system of neural transmission and signal transduction is a complicated, highly regulated cascade of biochemical events. Growing evidence suggests that receptor-G-protein coupling may be involved in both the pathogenesis and treatment of mood disorders. Our knowledge concerning the basic mechanisms underlying the phenomenon of desensitization, internalization, downregulation and resensitization of the G-protein-coupled receptor has been advanced during the last decade. The present review discusses the possible involvement of regulators of G-protein-coupled receptor-G-protein coupling: βarrestins, G-protein-coupled receptor kinases and phosducin-like proteins, as well as βarrestins alternative signaling events, in the pathophysiology, diagnosis and treatment monitoring of mood disorders and in the mechanism of action of antidepressant medications. [ABSTRACT FROM AUTHOR]

1 Cyclic AMP formation has consistently been reported to be desensitized in various tissues including heart of animal models of end-stage renal failure (ESRF). In contrast, reports on desensitization of cAMP formation in ESRF patients remain contradictory. Whether this discrepancy results from a difference between human ESRF and its animal models or from the use of circulating blood cells in the human and various solid tissues in the animal studies, remains unclear. Therefore, we performed three studies with heart and platelets of ESRF patients undergoing haemodialysis or continuous ambulatory peritoneal dialysis and age- and gender-matched controls with normal renal function (n = 11–13 each).2 In platelets from haemodialysis patients adenylyl cyclase activity in response to receptor-dependent and -independent agonists was reduced by≈30%, and this could be explained by an alteration at the level of adenylyl cyclase itself. However, no such desensitization was seen in platelets from peritoneal dialysis patients.3 In hearts from ESRF patients undergoing haemodialysis,β-adrenoceptor density and subtype distribution, cAMP formation in response to theβ-adrenoceptor agonist isoprenaline or various receptor-independent stimuli, were very similar to those in control patients but activity of G-protein-coupled receptor kinase was increased by≈20%.4 We conclude that conflicting reports on the desensitization of cAMP formation between ESRF patients and ESRF animal models are not explained by the use of solid tissues in animal studies vs. circulating blood cells in patient studies. Rather desensitization of cAMP formation seems to be a less consistent feature of human ESRF than of its animal models. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. G-protein-coupled receptor kinases (GRKs) play a key role in agonist-induced desensitisation of G-protein-coupled receptors (GPCRs) that are involved in metabolic regulation and glucose homeostasis. Our aim was to examine whether small peptides derived from the catalytic domain of GRK2 and -3 would ameliorate Type 2 diabetes in three separate animal models of diabetes. Methods. Synthetic peptides derived from a kinase-substrate interaction site in GRK2/3 were initially screened for their effect on in vitro melanogenesis, a GRK-mediated process. The most effective peptides were administered intraperitoneally, utilising a variety of dosing regimens, to Psammomys obesus gerbils, Zucker diabetic fatty (ZDF) rats, or db/db mice. The metabolic effects of these peptides were assessed by measuring fasting and fed blood glucose levels and glucose tolerance. Results. Two peptides, KRX-683107 and KRX-683124, significantly reduced fed-state blood glucose levels in the diabetic Psammomys obesus. In animals treated with KRX-683124 at a dose of 12.5 mg/kg weekly for 7 weeks, ten of eleven treated animals responded with mean blood glucose significantly lower than controls (4.7±0.4 vs 16.8±0.8 mmol/l, p≤0.0001). Significant reductions in blood glucose compared with controls were also seen in ZDF rats administered KRX-683124 and in db/db mice, which had significantly reduced fasting and 2-hour postprandial glucose levels after the treatment. Conclusions/interpretation. Sequence-based peptides derived from GRK2/3 have an antidiabetic effect demonstrated in three different animal models of Type 2 diabetes. By modulating GRK2/3 activity, these peptides enhance GPCR-initiated signal transduction, resulting in improved glucose homeostasis. Sequence-based peptide modulation of GRK could prove useful in the treatment of Type 2 diabetes. [ABSTRACT FROM AUTHOR]

We used the inositol 1,4,5-trisphosphate (IP3 ) biosensor, the pleckstrin homology (PH) domain of PLCδ1 (phospholipase C) tagged with enhanced green fluorescent protein (eGFP-PHPLCδ), to examine muscarinic acetylcholine (mACh) receptor regulation of phospholipase C/IP3 signaling in intact single hippocampal neurons in "real time." Initial experiments produced a pharmacological profile consistent with the presence of a predominant M1 mACh receptor population coupled to the IP3 response. To investigate M1 mACh receptor regulation, neurons were stimulated with approximate EC50 concentrations of the mACh receptor agonist methacholine before (R1) and after (R2) a short (60 sec) exposure to a high concentration of agonist. This resulted in a marked attenuation in the R2 relative to R1 response. Inhibition of endogenous GRK6 (G-protein-coupled receptor kinase) activity, by the introduction of catalytically inactive K215RGRK6, partially reversed the attenuation of agonist-induced responsiveness, whereas overexpression of wild-type GRK6 increased receptor desensitization. Manipulation of endogenous GRK2 activity through introduction of either wild-type or catalytically inactive GRK2 (K220RGRK2) almost completely inhibited agonist-stimulated IP3 production, implying a phosphorylation-independent regulation of M1 mACh receptor signaling, most probably mediated by a GRK2 N-terminal RGS-like (regulator of G-protein signaling) domain interaction with GTP-bound Gαq/11. Together, our data suggest a role for both phosphorylation-dependent and -independent regulation of M1 mACh receptors in hippocampal neurons. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in β-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in βIII -tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of β-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of βI -tubulin (393–445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length β-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2. [ABSTRACT FROM AUTHOR]

Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin (Rec), a retina-specific Ca2+ binding protein, and its aberrant expression in tumor cells lead to the retinal degeneration. To elucidate functional roles of the aberrantly expression in cancer cells, we performed immunoprecipitation using anti-human Rec mAb. We observed co-precipitation of G-protein-coupled receptor kinases (GRKs) and caveolin-1 with Rec from cell lysates of 293 or SSTW cells. Immunocytochemistry revealed that immunoreactivities toward Rec within the cancer cells were almost identical to those toward GRKs and caveolin-1. The present data strongly suggest that aberrantly expressed Rec should be involved in the GRK-dependent cellular regulation in cancer cells. [Copyright &y& Elsevier]

Tubulin was found to bind to a glutathione S-transferase fusion protein containing the carboxy-terminal domain of GTP-binding-protein-coupled receptor kinase 2 (GRK2) (residues 467-689), which is known to contain a pleckstrin homology site and to bind GTP-binding protein βγ subunits. The binding of tubulin to the fusion protein was not affected by GTP-binding protein βγ subunits, indicating that tubulin and βγ subunits bind GRK2 independently. Western-blotting analysis with anti-GRK2 Ig indicated that GRK2 was copurified with tubulin through the polymerization-depolymerization procedure. Tubulin was phosphorylated by GRK2, in contrast with the facts that the known substrates of GRK2 are restricted to activated forms of GTP-binding-protein-coupled receptors and that tubulin is a poor substrate for most kinases. GRK2 did not phosphorylate microtubule-associated proteins (MAPs), under conditions where MAPs were well phosphorylated by endogenous kinases copurified with tubulin. The Km for tubulin was estimated to be 3 μM, and 1.3 mol phosphate/tubulin dimer was incorporated. The phosphorylation of tubulin was stimulated by βγ subunits and agonist-bound muscarinic receptors. Phosphorylated tubulin could be polymerized into microtubules, and polymerized tubulin was also phosphorylated by GRK2. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptors are deactivated or desensitized by phosphorylation by respective G-protein-coupled receptor kinases (GRKs). In zebrafish rod and cone photoreceptor cells, four orthologous GRKs are expressed participating in the deactivation of rod and cone opsins. An important feature of GRKs in general is the consensus sites for lipid modification, which would allow the posttranslational attachment of isoprenoids facilitating membrane association and enzymatic performance. Because direct proof is missing for isoprenoid modification of zebrafish GRKs, we used a semichemical approach to study the incorporation of a farnesyl moiety into a GRK and its cellular consequences. The approach involves organic synthesis of a functionalized farnesyl derivative that is suitable for a subsequent alkyne-azide cycloaddition (click reaction). For this purpose, zebrafish GRK was expressed in HEK293 cells and modified in situ with the synthetic farnesyl moiety. Successful farnesylation by an endogenous farnesyltransferase was detected by immunoblotting and immunocytochemistry using a biotin-streptavidin-coupled assay and ligation with a fluorescence dye, respectively. Immunocytochemical detection of farnesylated GRK in different cell compartments indicates the applicability of the approach for studying the transport of cellular components.

Hedgehog (Hh) signaling plays a key role in the development and maintenance of animal tissues. This signaling is mediated by the atypical G protein-coupled receptor (GPCR) Smoothened (Smo). Smo activation leads to signaling through several well-characterized effectors to activate Hh target gene expression. Recent studies have implicated activation of the heterotrimeric G protein subunit Gα i and the subsequent decrease in cellular cAMP levels in promoting the Hh response in flies and mammals. Although Hh stimulation decreases cAMP levels in some insect cell lines, here using a bioluminescence resonance energy transfer (BRET)-based assay we found that this stimulation had no detectable effect in Drosophila S2-R+ cells. However, we observed an unexpected and significant Gα s -dependent increase in cAMP levels in response to strong Smo activation in Smo-transfected cells. This effect was mediated by Smo's broadly conserved core, and was specifically activated in response to phosphorylation of the Smo C-terminus by GPCR kinase 2 (Gprk2). Genetic analysis of heterotrimeric G protein function in the developing Drosophila wing revealed a positive role for cAMP in the endogenous Hh response. Specifically, we found that mutation or depletion of Gα s diminished low-threshold Hh responses in Drosophila , whereas depletion of Gα i potentiated them (in contrast to previous findings). Our analysis suggested that regulated cAMP production is important for controlling the sensitivity of cellular responses to Hh in Drosophila ., (© 2018 Praktiknjo et al.)

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 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 (Src pY416 ) is co-expressed in Exo with phosphorylated FAK (FAK pY861 ), 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, Src pY416 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. J. Cell. Biochem. 118: 66-73, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

G-protein-coupled receptor kinases (GRKs) play a conserved role in Hedgehog (Hh) signaling. In several systems, GRKs are required for efficient Hh target gene expression. Their principal target appears to be Smoothened (Smo), the intracellular signal-generating component of the pathway and a member of the G-protein-coupled receptor (GPCR) protein family. In Drosophila, a GRK called Gprk2 is needed for internalization and downregulation of activated Smo, consistent with the typical role of these kinases in negatively regulating GPCRs. However, Hh target gene activation is strongly impaired in gprk2 mutant flies, indicating that Gprk2 must also positively regulate Hh signaling at some level. To investigate its function in signaling, we analyzed several different readouts of Hh pathway activity in animals or cells lacking Gprk2. Surprisingly, although target gene expression was impaired, Smo-dependent activation of downstream components of the signaling pathway was increased in the absence of Gprk2. This suggests that Gprk2 does indeed play a role in terminating Smo signaling. However, loss of Gprk2 resulted in a decrease in cellular cAMP concentrations to a level that was limiting for Hh target gene activation. Normal expression of target genes was restored in gprk2 mutants by stimulating cAMP production or activating the cAMP-dependent Protein kinase A (Pka). Our results suggest that direct regulation of Smo by Gprk2 is not absolutely required for Hh target gene expression. Gprk2 is important for normal cAMP regulation, and thus has an indirect effect on the activity of Pka-regulated components of the Hh pathway, including Smo itself. [ABSTRACT FROM AUTHOR]

Objective: Recent evidence suggests G-protein-coupled receptor-2-interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown., Approach and Results: In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250-420) of GIT1 is required for GIT1-cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2-mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells., Conclusion: Our data demonstrated that a GIT1-cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.

α-Synuclein is the major component of Lewy bodies. α-Synuclein phosphorylated at Ser 129 (Phospho-α-Syn) is the most common synuclein modification observed in Parkinson's disease pathology and transgenic animal models. Polo-like kinase 2 (PLK2) was previously proposed as an important kinase in α-synuclein phosphorylation at Ser129. To better understand the role of PLK2 in α-synuclein phosphorylation in vivo, we further evaluated the effect of PLK2 genetic knockdown and pharmacological inhibition on Phospho-α-Syn levels in different brain regions of PLK2 knockout (KO), heterozygous (Het) and wild-type (WT) mice. Whereas PLK2 knockdown had no effect on Total-α-synuclein brain levels, it resulted in a gene-dosage dependent, albeit incomplete, reduction of endogenous Phospho-α-Syn levels in all brain regions investigated. No compensatory induction of other α-synuclein kinases (PLK3, casein kinase-2, G-protein-coupled receptor kinase 5 (GRK5) and GRK6) was observed at the mRNA level in the PLK2 KO mouse brain. To determine whether increased activity of another PLK family member is responsible for the residual Phospho-α-Syn levels in the PLK2 KO mouse brain, the pan-PLK inhibitor BI 2536 was tested in PLK2 KO mice. Whereas BI 2536 reduced Phospho-α-Syn levels in WT mice, it did not further reduce the residual endogenous Phospho-α-Syn levels in PLK2 KO and Het mice, suggesting that a kinase other than PLK1-3 accounts for the remaining PLK inhibitor-resistant pool in the mouse brain. Moreover, PLK3 KO in mice had no effect on both Total- and Phospho-α-Syn brain levels. These results support a significant role for a PLK kinase in phosphorylating α-synuclein at Ser129 in the brain, and suggest that PLK2 is responsible for this activity under physiological conditions., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.