G-protein Receptor Kinase Research Articles

Ectopic expression of cone‐specific G‐protein‐coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses

To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7(giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods.

Recovery from μ-Opioid Receptor Desensitization after Chronic Treatment with Morphine and Methadone

Chronic treatment with morphine results in a decrease in μ-opioid receptor sensitivity, an increase in acute desensitization, and a reduction in the recovery from acute desensitization in locus ceruleus neurons. With acute administration, morphine is unlike many other opioid agonists in that it does not mediate robust acute desensitization or induce receptor trafficking. This study compares μ-opioid receptor desensitization and trafficking in brain slices taken from rats treated for 6-7 d with a range of doses of morphine (60, 30, and 15 mg · kg(-1) · d(-1)) and methadone (60, 30, and 5 mg · kg(-1) · d(-1)) applied by subcutaneous implantation of osmotic minipumps. Mice were treated with 45 mg · kg(-1) · d(-1). In morphine-treated animals, recovery from acute [Met](5)enkephalin-induced desensitization and receptor recycling was diminished. In contrast, recovery and recycling were unchanged in slices from methadone-treated animals. Remarkably the reduced recovery from desensitization and receptor recycling found in slices from morphine-treated animals were not observed in animals lacking β-arrestin-2. Furthermore, pharmacological inhibition of G-protein receptor kinase 2 (GRK2), although not affecting the ability of [Met](5)enkephalin to induce desensitization, acutely reversed the delay in recovery from desensitization produced by chronic morphine treatment. These results characterize a previously unidentified function of the GRK/arrestin system in mediating opioid regulation in response to chronic morphine administration. They also suggest that the GRK/arrestin system, rather than serving as a primary mediator of acute desensitization, controls recovery from desensitization by regulating receptor reinsertion to the plasma membrane after chronic treatment with morphine. The sustained GRK/arrestin-dependent desensitization is another way in which morphine and methadone are distinguished.

Endocytosis in the mouse oocyte and its contribution to cAMP signaling during meiotic arrest

Mammalian oocytes are arrested at prophase I of meiosis until a preovulatory surge of LH stimulates them to resume meiosis. Prior to the LH surge, high levels of cAMP within the oocyte maintain meiotic arrest; this cAMP is generated in the oocyte through the activity of the constitutively active, G(s)-coupled receptor, G-protein-coupled receptor 3 (GPR3) or GPR12. Activated GPRs are typically targeted for desensitization through receptor-mediated endocytosis, but a continuously high level of cAMP is needed for meiotic arrest. The aim of this study was to examine whether receptor-mediated endocytosis occurs in the mouse oocyte and whether this could affect the maintenance of meiotic arrest. We found that constitutive endocytosis occurs in the mouse oocyte. Inhibitors of receptor-mediated endocytosis, monodansylcadaverine and dynasore, inhibited the formation of early endosomes and completely inhibited spontaneous meiotic resumption. A red fluorescent protein-tagged GPR3 localized in the plasma membrane and within early endosomes in the oocyte, demonstrating that GPR3 is endocytosed. However, overexpression of G-protein receptor kinase 2 and β-arrestin-2 had only a modest effect on stimulating meiotic resumption, suggesting that these proteins do not play a major role in GPR3 endocytosis. Inhibition of endocytosis elevated cAMP levels within oocytes, suggesting that there is an accumulation of GPR3 at the plasma membrane. These results show that endocytosis occurs in the oocyte, leading to a decrease in cAMP production, and suggest that there is a balance between cAMP production and degradation in the arrested oocyte that maintains cAMP levels at an appropriate level during the maintenance of meiotic arrest.

Role of beta-arrestin 2 downstream of dopamine receptors in the basal ganglia

Multifunctional scaffolding protein beta-arrestins (βArr) and the G protein-receptor kinases are involved in the desensitization of several G protein-coupled receptors (GPCR). However, arrestins can also contribute to GPCR signaling independently from G proteins. In this review, we focus on the role of βArr in the regulation of dopamine receptor functions in the striatum. First, we present in vivo evidence supporting a role for these proteins in the regulation of dopamine receptor desensitization. Second, we provide an overview of the roles of βArr2 in the regulation of extracellular-signal-regulated kinases/MAP kinases and Akt/GSK3 signaling pathways downstream of the D1 and D2 dopamine receptors. Thereafter, we examine the possible involvement of βArr-mediated signaling in the action of dopaminergic drugs used for the treatment of mental disorders. Finally, we focus on different potential cellular proteins regulated by βArr-mediated signaling which could contribute to the regulation of behavioral responses to dopamine. Overall, the identification of a cell signaling function for βArr downstream of dopamine receptors underscores the intricate complexity of the intertwined mechanisms regulating and mediating cell signaling in the basal ganglia. Understanding these mechanisms may lead to a better comprehension of the several roles played by these structures in the regulation of mood and to the development of new psychoactive drugs having better therapeutic efficacy.

Real-time imaging of leukotriene B₄ mediated cell migration and BLT1 interactions with β-arrestin.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins(1-2). Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of β-arrestins(3). β-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization (4-6). Leukotriene B(4;) (LTB(4;)) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB(4;) receptor 1 (BLT1; a high affinity receptor) and LTB(4;) receptor 2 (BLT2; a low affinity receptor)(7-9). The LTB(4;)-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis(10-17). The current paper describes the methodologies developed to monitor LTB(4;)-induced leukocyte migration and the interactions of BLT1 with β-arrestin and , receptor translocation in live cells using microscopy imaging techniques(18-19). Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described (20-21). These cells were tested in live cell imaging methods to demonstrate LTB(4;) induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and β-arrestin1 tagged with green fluorescent protein (β-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines(18-19). The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Lysophosphatidylcholines activate G2A inducing Gαi-1-/Gαq/11- Ca2+ flux, Gβγ-Hck activation and clathrin/β-arrestin-1/GRK6 recruitment in PMNs

Lyso-PCs (lysophosphatidylcholines) are a mixture of lipids that accumulate during storage of cellular blood components, have been implicated in TRALI (transfusion-related acute lung injury) and directly affect the physiology of neutrophils [PMNs (polymorphonuclear leucocytes)]. Because the G2A receptor, expressed on PMNs, has been reported to recognize lyso-PCs, we hypothesize that lyso-PC activation of G2A causes the increases in cytosolic Ca²(+) via release of G(α) and G(βγ) subunits, kinase activation, and the recruitment of clathrin, β-arrestin-1 and GRK6 (G-protein receptor kinase 6) to G2A for signal transduction. PMNs were isolated by standard techniques, primed with lyso-PCs for 5-180 s, and lysed for Western blot analysis, immunoprecipitation or subcellular fractionation, or fixed and smeared on to slides for digital microscopy. The results demonstrated that lyso-PCs cause rapid activation of the G2A receptor through S-phosphorylation and internalization resulting in G(αi)₋₁ and G(αq/)₁₁ release leading to increases in cytosolic Ca²(+), which was inhibited by an antibody to G2A or intracellular neutralization of these subunits. Lyso-PCs also caused the release of the G(βγ) subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr⁴¹¹). Moreover, G2A recruited clathrin, β-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and β-arrestin-1 both propagates and terminates signals. We conclude that lyso-PC activation of G2A caused release of G(αi)₋₁, G(αq/)₁₁ and G(βγ), resulting in cytosolic Ca²(+) flux, Hck activation, and recruitment of clathrin, β-arrestin-1 and GRK6.

Ca 2+ Cycling and New Therapeutic Approaches for Heart Failure

Received July 2, 2009; accepted October 5, 2009. Heart failure (HF) is a major health problem in Western countries. Despite significant progress in pharmacological and device-based treatment, the disease burden imposed continues to increase, particularly as the population ages. HF incidence approaches 10 per 1000 after age 65 years.1 Congestive HF is the final consequence of diverse cardiovascular disorders, including atherosclerosis, cardiomyopathy, and hypertension. Described as a complex pathophysiological syndrome that involves interactions of the circulatory, neurohormonal, and renal systems, HF is first a disease of the myocardium, although it soon induces defects in other systems. Current treatments for HF, focused on blocking neurohormonal pathways, improve survival, but they do not halt the progression of HF. Late-stage HF has a poor prognosis, and therapeutic options are limited. Faced with these challenges, researchers are exploring novel therapeutic options. Chronic HF is associated with increased sympathetic outflow, which may be compensatory early on, but long-term neurohormonal activation induces significant damage to the heart; in addition, it results in multiple alterations in the β-adrenergic receptor (β-AR) signaling cascade, including receptor downregulation, upregulation of receptor kinases, and increased inhibitory G-protein function.2 The amplitude and velocity of Ca2+ cycling are regulated by a dynamic balance of phosphorylation and dephosphorylation through kinases and phosphatases. Activation of β-ARs stimulates cAMP production and results in protein kinase A (PKA) phosphorylation of key regulators of excitation-contraction coupling, such as L-type Ca2+ channels, phospholamban, troponin I, ryanodine receptors (RyR), myosin-binding protein C, and protein phosphatase inhibitor-1 (I-1; Figure), which leads to increased amplitude and velocity of Ca2+ cycling and increased contractility on a beat-to-beat basis.3 Protein phosphatases PP1 and PP2A counterbalance phosphorylation of these proteins. There is clear evidence that alterations in sarcoplasmic reticulum (SR) Ca2+ cycling are a component of the impaired …

Differences in the C‐terminus contribute to variations in trafficking between rat and human 5‐HT2A receptor isoforms: identification of a primate‐specific tripeptide ASK motif that confers GRK‐2 and β arrestin‐2 interactions

Internalization and recycling of G-protein coupled receptors are important cellular processes regulating receptor function. These are receptor-subtype and cell type-specific. Although important, trafficking variations between receptor isoforms of different species has received limited attention. We report here, differences in internalization and recycling between rat and human serotonin 2A receptor (5-HT(2A)R) isoforms expressed in human embryonic kidney 293 cells in response to serotonin. Although the human and rat 5-HT(2A)Rs differ by only a few amino acids, the human receptor takes longer to recycle to the cell surface after internalization, with the additional involvement of beta arrestin-2 and G-protein receptor kinase 2. The interaction of beta arrestin-2 with the human receptor causes the delay in recycling and is dependent on a primate-specific ASK motif present in the C-terminus of the receptor. Conversion of this motif to NCT, the corresponding sequence present in the rat isoform, results in the human isoform trafficking like the rat receptor. Replacing the serine 457 with alanine in the ASK motif of human isoform resulted in faster recycling, although with continued arrestin-dependent internalization. This study establishes significant differences between the two isoforms with important implications in our understanding of the human 5-HT(2A)R functions; and indicates that extrapolating results from non-human receptor isoforms to human subtypes is not without caveats.

Cardiovascular hyporesponsiveness in sepsis is associated with G-protein receptor kinase expression via a nitric oxide-dependent mechanism

Septic shock is characterized by cardiac collapse and decreased peripheral resistance due to systemic resistance vessel dilatation, generally induced by large nitric oxide (NO). G-protein-coupled receptor (GPCR) kinases (GRKs), specific kinases interacting with GPCR proteins, induce receptor phosphorylation and thereby signal desensitization in the continuing agonist presence. Then, an increased expression of GRKs could augment adrenergic receptor desensitization and in turn reduce cardiovascular responses. Thus, we hypothesized that the hyporesponsiveness observed in sepsis could result from signal adrenergic receptor desensitization mediated by GRK2 via a NO-dependent mechanism.

Tyrosine Phosphorylation of Kir3 following κ-Opioid Receptor Activation of p38 MAPK Causes Heterologous Desensitization

Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K(ir)3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K(ir)3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wild-type mice, but not in mice lacking the kappa-opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K(ir)3.1-ir. The U50,488-induced increase in pY12-K(ir)3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K(ir)3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K(ir)3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K(ir)3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K(ir)3.1-ir; and the heterologous desensitization of K(ir)3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K(ir)3.1 and channel inhibition through a GRK3-, p38 MAPK- and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.

Assays for S -Nitrosothiols and S -Nitrosylated Proteins and Mechanistic Insights Into Cardioprotection

Gender differences in the incidence of cardiovascular disease may be ascribed at least in part to the protective effects of estrogen through both long-term and rapid (“nongenomic”) actions.1 Nitric oxide (NO), generated by endogenous cardiac NO synthases (NOS; NOS1 or neuronal NOS [nNOS], NOS2 or inducible NOS, NOS3 or endothelial NOS), plays a major role in both normal cardiac physiology and cardioprotection (particularly myocardial ischemia/reperfusion injury; see the Figure),2 and the article by Lin et al3 in this issue of Circulation contributes to growing evidence that the cardioprotective functions of estrogen are conveyed in significant part by NO. A rapidly expanding body of studies indicates that NO acts in most cellular contexts largely through the covalent modification of protein Cys thiols (to generate an S -nitroso [SNO]-protein, designated S -nitrosylation),4 and recent analyses using a new generation of analytical approaches (see the Table) both confirm original measurements of SNO-proteins that have been long-standing sources of controversy in the field and point to important roles for S -nitrosylation in NO-derived cardioprotection (see the Figure).3,5,6 Figure. NO-based mechanisms for preconditioning in ischemia/reperfusion injury. Accumulating evidence suggests a role for NO/SNO in estrogen and adrenergic receptor–mediated and statin-induced preconditioning in ischemia/reperfusion injury. Protein S -nitrosylation, resulting from increased NOS expression and activity and altered subcellular localization, appears to be a principal mediator of these effects. View this table: Table. Methodologies for the Detection and Absolute or Relative Quantification of SNO-Proteins and Other S -Nitrosothiols Article see p 245 In the myocardium, endothelial NOS is associated primarily with sarcolemmal caveolae and perhaps β-arrestin, and endothelial NOS–derived NO influences β-adrenergic receptor stimulation of myocardial contractility,7 at least in part through S -nitrosylation and inhibition of the L-type Ca2+ channel5 and G-protein receptor kinase 2.8 In contrast, nNOS …

Clinical and Genetic Modifiers of Long-Term Survival in Heart Failure

This study sought to identify genetic modifiers of beta-blocker response and long-term survival in heart failure (HF). Differences in beta-blocker treatment effect between Caucasians and African Americans with HF have been reported. This was a prospective cohort study of 2,460 patients (711 African American, 1,749 Caucasian) enrolled between 1999 and 2007; 2,039 patients (81.7%) were treated with a beta-blocker. Each was genotyped for beta1-adrenergic receptor (ADRB1) Arg389>Gly and G-protein receptor kinase 5 (GRK5) Gln41>Leu polymorphisms, which are more prevalent among African Americans than Caucasians. The primary end point was survival time from HF onset. There were 765 deaths during follow-up (median 46 months). beta-blocker treatment increased survival in Caucasians (log-rank p = 0.00038) but not African Americans (log-rank p = 0.327). Among patients not taking beta-blockers, ADRB1 Gly389 was associated with decreased survival in Caucasians (hazard ratio [HR]: 1.98, 95% confidence interval [CI]: 1.1 to 3.7, p = 0.03) whereas GRK5 Leu41 was associated with improved survival in African Americans (HR: 0.325, CI: 0.133 to 0.796, p = 0.01). African Americans with ADRB1 Gly389Gly GRK5 Gln41Gln derived a similar survival benefit from beta-blocker therapy (HR: 0.385, 95% CI: 0.182 to 0.813, p = 0.012) as Caucasians with the same genotype (HR: 0.529, 95% CI: 0.326 to 0.858, p = 0.0098). These data show that differences caused by beta-adrenergic receptor signaling pathway gene polymorphisms, rather than race, are the major factors contributing to apparent differences in the beta-blocker treatment effect between Caucasians and African Americans; proper evaluation of treatment response should account for genetic variance.

Repeated Bouts of Moderate-Intensity Aerobic Exercise Reduce Airway Reactivity in a Murine Asthma Model

We have reported that moderate-intensity aerobic exercise training attenuates airway inflammation in mice sensitized/challenged with ovalbumin (OVA). The current study determined the effects of repeated bouts of aerobic exercise at a moderate intensity on airway hyperresponsiveness (AHR) in these mice. Mice were sensitized/challenged with OVA or saline and exercised at a moderate intensity 3 times/week for 4 weeks. At protocol completion, mice were analyzed for changes in AHR via mechanical ventilation. Results show that exercise decreased total lung resistance 60% in OVA-treated mice as compared with controls; exercise also decreased airway smooth muscle (ASM) thickness. In contrast, exercise increased circulating epinephrine levels 3-fold in saline- and OVA-treated mice. Because epinephrine binds beta(2)-adrenergic receptors (AR), which facilitate bronchodilatation, the role of beta(2)-AR in exercise-mediated improvements in AHR was examined. Application of the beta(2)-AR antagonist butoxamine HCl blocked the effects of exercise on lung resistance in OVA-treated mice. In parallel, ASM cells were examined for changes in the protein expression of beta(2)-AR and G-protein receptor kinase-2 (GRK-2); GRK-2 promotes beta(2)-AR desensitization. Exercise had no effect on beta(2)-AR expression in ASM cells of OVA-treated mice; however, exercise decreased GRK-2 expression by 50% as compared with controls. Exercise also decreased prostaglandin E(2) (PGE(2)) production 5-fold, but had no effect on E prostanoid-1 (EP1) receptor expression within the lungs of OVA-treated mice; both PGE(2) and the EP1 receptor have been implicated in beta(2)-AR desensitization. Together, these data indicate that moderate-intensity aerobic exercise training attenuates AHR via a mechanism that involves beta(2)-AR.

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of α2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged alpha(2A)-adrenergic receptor (AR) expression was driven by the endogenous mouse alpha(2A)AR gene locus. Exploiting this mouse line, we evaluated alpha(2A)AR trafficking and alpha(2A)AR-mediated inhibition of Ca(2+) currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the alpha(2A)AR subtype. We discovered a more rapid desensitization of Ca(2+) current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of alpha(2A)AR evoked by clonidine than by guanfacine. Clonidine-induced alpha(2A)AR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of alpha(2A)AR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-alpha(2A)AR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

Decreased GRK3 but not GRK2 expression in frontal cortex from bipolar disorder patients

Overactivation of G-protein-mediated functions and altered G-protein regulation have been reported in bipolar disorder (BD) brain. Further, drugs effective in treating BD are reported to up-regulate expression of G-protein receptor kinase (GRK) 3 in rat frontal cortex. We therefore hypothesized that some G-protein subunits and GRK levels would be reduced in the brain of BD patients. We determined protein and mRNA levels of G-protein beta and gamma subunits, GRK2, and GRK3 in post-mortem frontal cortex from 10 BD patients and 10 age-matched controls by using immunoblots and real-time RT-PCR. There were statistically significant decreases in protein and mRNA levels of G-protein subunits beta and gamma and of GRK3 in BD brain but not a significant difference in the GRK2 level. Decreased expression of G-protein subunits and of GRK3 may alter neurotransmission, leading to disturbed cognition and behaviour in BD.

Mechanisms regulating the dopamine D1‐D2 receptor heteromeric signaling complex

We previously identified that co‐activation of a Gq‐linked heteromeric D1‐D2 dopamine receptor signaling complex results in phospholipase C (PLC)‐dependent intracellular Ca2+ release. We have demonstrated agonist specificity for the D1‐D2 triggered Ca2+ signal and the D1 stimulated cyclic AMP production with three D1 agonists, SKF83959, SKF81297, and SKF83822 that differentially activate either the adenylate cyclase (AC) or PLC pathway. Because the three agonists can activate different signaling pathways, we propose that they are effective pharmacological tools for assessing the mechanisms regulating the D1‐D2 signaling complex. Pretreatment of D1‐D2 expressing cells with each agonist lead to 60% desensitization of the Ca2+ signal within 5 minutes. Intriguingly, occupancy of the D1 binding pocket by SKF 83822 that could not activate the D1‐D2 complex still permitted desensitization of the Ca2+ response. Results from co‐application of the agonists with a D1 or D2 specific antagonist, suggest the desensitization elicited by each agonist is through selective occupancy of the D1 receptor. Furthermore, over‐expression of wild type G‐protein receptor kinase 2 (GRK2) and its catalytically inert construct, but not its regulator of G‐protein signaling (RGS) domain impaired construct, decreased the Ca2+ signal, suggesting the RGS domain of GRK2 is involved in regulating the D1‐D2 receptor signaling cascade.This work is supported by the NIH National Institute on Drug Abuse

Novel pharmacotherapies to abrogate postinfarction ventricular remodeling

Ventricular remodeling after myocardial infarction is defined as progressive chamber dilation and wall thinning, which leads to functional compromise. Remodeling is mediated by active processes of inflammation, fibrosis, and cardiomyocyte dropout over the weeks and months after infarction, and, therefore, provides a large temporal therapeutic window. In experimental models, interruption of molecular and physiological pathways that contribute to cardiomyocyte loss, and the resulting unfavorable ventricular geometry, can abrogate remodeling and prevent or improve heart failure. Remodeling is multifactorial and involves several parallel cellular pathways, which means many potential therapeutic targets exist. Of late, much attention has been given to the development of cell-based therapies; however, the abundant, promising pharmacotherapeutic developments should not be overlooked. This Review examines developments in pharmacological treatment of ventricular remodeling in preclinical models of myocardial infarction-specifically, disruption of the renin-angiotensin-aldosterone system through direct renin inhibition and blockade of aldosterone synthesis and/or uptake, enhancement of endothelial nitric oxide synthase synthesis, G-protein receptor kinase inhibition, administration of erythropoietin, and interruption of apoptosis-and highlights the challenge of translating these successes to treatment of human disease. Therapeutic targeting of multiple organ systems involved in recovery after myocardial infarction might prove to be the best approach to improve patients' cardiac outcome.

Sonic Hedgehog signaling in astrocytes is dependent on p38 mitogen‐activated protein kinase and G‐protein receptor kinase 2

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh-regulated gene targets, Gli-1 and Patched in a cyclopamine- and forskolin-sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular-signal-regulated kinases or c-jun N-terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh-dependent up-regulation of Gli-1, inter-alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin-like growth factor 2 or a novel Shh target, membrane-associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively-active mutant we show that Shh signaling to p38 MAPK and subsequent Gli-1 transcription requires G-protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G-protein receptor kinase 2-dependent p38 MAPK activity in regulating Shh-mediated gene transcription in astrocytes.

GRK mythology: G-protein receptor kinases in cardiovascular disease

G-protein receptor kinases (GRKs) are indispensable for terminating signaling of G-protein coupled receptors (GPCR) through receptor desensitization and downregulation. Increased neurohormone levels in heart failure and the adverse consequences of constant neurohormonal stimulation suggest an important protective role for mechanisms that desensitize neurohormone receptor responses. For that reason, GRK2, the first GRK identified in the heart, has been extensively studied in heart failure, cardiac hypertrophy, and myocardial infarction. However, our understanding of the roles of GRKs in general, and the differential effects of cardiac receptor phosphorylation by individual cardiac-expressed GRKs, have evolved considerably in the last few years. Here, recent developments are reviewed, with an emphasis on novel GRK functions and signaling pathways.

Altered Sensitivity to Rewarding and Aversive Drugs in Mice with Inducible Disruption of cAMP Response Element-Binding Protein Function within the Nucleus Accumbens

The transcription factor cAMP response element-binding protein (CREB) within the nucleus accumbens (NAc) plays an important role in regulating mood. In rodents, increased CREB activity within the NAc produces depression-like signs including anhedonia, whereas disruption of CREB activity by expression of a dominant-negative CREB (mCREB, which acts as a CREB antagonist) has antidepressant-like effects. We examined how disruption of CREB activity affects brain reward processes using intracranial self-stimulation (ICSS) and inducible bitransgenic mice with enriched expression of mCREB in forebrain regions including the NAc. Mutant mice or littermate controls were prepared with lateral hypothalamic stimulating electrodes, and trained in the ICSS procedure to determine the frequency at which the stimulation becomes rewarding (threshold). Inducible expression of mCREB did not affect baseline sensitivity to brain stimulation itself. However, mCREB-expressing mice were more sensitive to the rewarding (threshold-lowering) effects of cocaine. Interestingly, mCREB mice were insensitive to the depressive-like (threshold-elevating) effects of the kappa-opioid receptor agonist U50,488. These behavioral differences were accompanied by decreased mRNA expression of G-protein receptor kinase-3 (GRK3), a protein involved in opioid receptor desensitization, within the NAc of mCREB mice. Disruption of CREB or GRK3 activity within the NAc specifically by viral-mediated gene transfer enhanced the rewarding impact of brain stimulation in rats, establishing the contribution of functional changes within this region. Together with previous findings, these studies raise the possibility that disruption of CREB in the NAc influences motivation by simultaneously facilitating reward and reducing depressive-like states such as anhedonia and dysphoria.