cAMP Formation Research Articles

Smurf1 regulates lung cancer cell growth and migration through interaction with and ubiquitination of PIPKIγ.

Type Iγ phosphatidylinositol phosphate kinase (PIPKIγ), a phospholipid kinase generating PIP2, is positively expressed in breast cancer tissues, which correlates intimately with the progression of patients. However, little is known about the expression level of PIPKIγ in patients with other cancer types as well as their underlying regulation mechanisms. Here, we report that PIPKIγ is highly expressed in lung cancer tissues and its expression level is critical for lung cancer cell proliferation, which may serve as a prognostic marker for lung cancer patients. Meanwhile, we show that E3 ubiquitin ligase Smurf1 directly interacts with PIPKIγ and targets PIPKIγ for ubiquitination and degradation in lung cancer cells. Also, we discover that Smurf1 directly binds to the kinase domain of PIPKIγ via its C2 domain while Lysine 255 in PIPKIγ acts as the major ubiquitin acceptor site for Smurf1. In addition, we demonstrate that the phosphorylation mimicking mutant of Smurf1, Smurf1 T306D, prevents PIPKIγi2 from ubiquitination and subsequent degradation similar to the effect of forskolin-potentiated cAMP formation, suggesting that Thr306 in Smurf1 is critical for its phosphorylation by PKA. Moreover, PKA-Smurf1-PIPKIγ signal transduction takes a significant part in lung cancer cell growth and in vivo tumorigenesis. Thus, we propose that the PKA-Smurf1-PIPKIγ pathway has an important role in pulmonary tumorigenesis and imposes substantial clinical impact on development of novel diagnostic markers and therapeutic targets for lung cancer treatment.

Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus Blastocladiella emersonii

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus Blastocladiella emersonii It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λmax = 390 nm; D380N, λmax = 506 nm) and one with red-shifted (D380E, λmax = 533 nm) absorption maxima relative to the wild-type protein (λmax = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.

Soluble Ectodomain of Neuroligin 1 Decreases Synaptic Activity by Activating Metabotropic Glutamate Receptor 2.

Synaptic cell adhesion molecules represent important targets for neuronal activity-dependent proteolysis. Postsynaptic neuroligins (NLs) form trans-synaptic complexes with presynaptic neurexins (NXs). Both NXs and NLs are cleaved from the cell surface by metalloproteases in an activity-dependent manner, releasing a soluble extracellular fragment and membrane-tethered C-terminal fragment. The cleavage of NL1 depresses synaptic transmission, but the mechanism by which this occurs is unknown. Metabotropic glutamate receptor 2 (mGluR2) are located primarily at the periphery of presynaptic terminals, where they inhibit the formation of cyclic adenosine monophosphate (cAMP) and consequently suppress the release of glutamate and decrease synaptic transmission. In the present study, we found that the soluble ectodomain of NL1 binds to and activates mGluR2 in both neurons and heterologous cells, resulting in a decrease in cAMP formation. In a slice preparation from the hippocampus of mice, NL1 inhibited the release of glutamate from mossy fibers that project to CA3 pyramidal neurons. The presynaptic effect of NL1 was abolished in the presence of a selective antagonist for mGluR2. Thus, our data suggest that the soluble extracellular domain of NL1 functionally interacts with mGluR2 and thereby decreases synaptic strength.

Catecholamines facilitate VEGF-dependent angiogenesis via β2-adrenoceptor-induced Epac1 and PKA activation.

Chronic stress has been associated with the progression of cancer and antagonists for β-adrenoceptors (βAR) are regarded as therapeutic option. As they are also used to treat hemangiomas as well as retinopathy of prematurity, a role of endothelial β2AR in angiogenesis can be envisioned. We therefore investigated the role of β2AR-induced cAMP formation by analyzing the role of the cAMP effector molecules exchange factor directly activated by cAMP 1 (Epac1) and protein kinase A (PKA) in endothelial cells (EC). Epac1-deficient mice showed a reduced amount of pre-retinal neovascularizations in the model of oxygen-induced retinopathy, which is predominantly driven by vascular endothelial growth factor (VEGF). siRNA-mediated knockdown of Epac1 in human umbilical vein EC (HUVEC) decreased angiogenic sprouting by lowering the expression of the endothelial VEGF-receptor-2 (VEGFR-2). Conversely, Epac1 activation by β2AR stimulation or the Epac-selective activator cAMP analog 8-p-CPT-2’-O-Me-cAMP (8-pCPT) increased VEGFR-2 levels and VEGF-dependent sprouting. Similar to Epac1 knockdown, depletion of the monomeric GTPase Rac1 decreased VEGFR-2 expression. As Epac1 stimulation induces Rac1 activation, Epac1 might regulate VEGFR-2 expression through Rac1. In addition, we found that PKA was also involved in the regulation of angiogenesis in EC since the adenylyl cyclase (AC) activator forskolin (Fsk), but not 8-pCPT, increased sprouting in Epac1-depleted HUVEC and this increase was sensitive to a selective synthetic peptide PKA inhibitor. In accordance, β2AR- and AC-activation, but not Epac1 stimulation increased VEGF secretion in HUVEC.Our data indicate that high levels of catecholamines, which occur during chronic stress, prime the endothelium for angiogenesis through a β2AR-mediated increase in endothelial VEGFR-2 expression and VEGF secretion.

The Catalytic α Subunit of cAMP‐Dependent Protein Kinase Regulates Formoterol‐Induced Gene Expression in BEAS‐2B Human Bronchial Epithelial Cells

RationaleThe β2‐adrenoceptor is a GPCR that classically couples to Gsα, the activation of adenylyl cyclases and the formation of cAMP. Through distinct signalling complexes often containing cAMP‐dependent protein kinase (PKA), cAMP mediates a myriad of functional responses including the regulation of gene expression. We have shown previously that in the BEAS‐2B human airway epithelial cell line, long acting β2‐adrenoceptor agonists (LABAs) induce cAMP response element (CRE) dependent transcription, which we suggest contributes to their therapeutic activity in obstructive lung diseases. In the current study we have extended those findings by identifying the PKA catalytic subunit responsible for mediating gene expression induced by the LABA, formoterol.MethodsMicroarray, RT‐PCR, western blotting and gene silencing were used to identify and implicate PKA catalytic subunits in the regulation of LABA‐induced gene expression. A CRE reporter construct stably transfected into BEAS‐2B cells was used for this purpose together with an assessment of CREB phosphorylation and an analysis of a panel of cAMP‐inducible genes (NR4A3, CRISPLD2, RGS2, NR4A2, CD200 and IL‐6).ResultsMicroarray‐based gene expression profiling indicated that BEAS‐2B cells express mRNA transcripts for PRKACA (Cα), PRKACB (Cβ) and PRKX (Cx) whereas PRKACG (Cg) was absent. These data were confirmed at the protein level by western blotting. siRNAs were optimized to achieve 80 to 90% knockdown of PRKACA, PRKACB and PRKX, which was reproduced at the protein level. Under these conditions, silencing PRKACA was associated with a significant (~50%) abrogation of formoterol‐induced CRE reporter activation, CREB phosphorylation and gene expression. No additional effect was seen when siRNAs that target PRKACB or PRKX were used in combination with those directed towards PRKACA.ConclusionsThese data show that Cα is an essential cAMP signalling component controlling formoterol‐induced gene transcription in BEAS‐2B cells. The incomplete inhibition produced under conditions of 80–90% Cα knockdown implies that this PKA catalytic subunit is present in significant excess of that required to maximally mediate cAMP‐induced gene expression. This is an initial first step at identifying cAMP signalling components (adenylyl cyclases, scaffold proteins, phosphodiesterases and effectors) that control LABA‐induced functional responses in airway structural cells. We propose that identifying these components and their assembly into multiprotein signalling complexes could provide new opportunities for the development of novel therapies for asthma and chronic obstructive pulmonary disease.Support or Funding InformationAlberta Lung Association (ALA)

Importance of adenylyl cyclase 6 in developing lithium‐induced nephrogenic diabetes insipidus

Nephrogenic diabetes insipidus (NDI) develops in approximately 50% of psychiatric patients being treated with lithium (Li+). The mechanism behind Li+‐induced NDI (Li‐NDI) is only poorly understood, but it occurs at least partly due to reduced aquaporin‐2 (AQP2) expression, leading to lower water reabsorption in collecting ducts (CD) of the kidney. A suggested mechanism inducing these effects is that Li+ inhibits adenylyl cyclase (AC) activity and thereby decreases cAMP levels and subsequently reduces AQP2 abundance and membrane targeting. In the present study, we tested the hypothesis that Li‐NDI would not develop in mice lacking AC6, the AC isoform responsible for the majority of vasopressin‐induced inner medullary (IM) cAMP formation. Total‐body AC6 knockout (AC6−/−) mice and newly developed connecting tubule/principal cell‐specific AC6 knockout mice (AC6loxloxCre) were studied. Similar to AC6−/− mice, the novel AC6loxloxCre mice had lower urine osmolality [mean ± SE (n): control 2363 ± 114 mmol/kg (17), AC6loxloxCre 1219 ± 49 mmol/kg (9); P < 0.05] and higher water intake [control 0.19 ± 0.01 g/g BW/24h (7), AC6loxloxCre 0.28 ± 0.02 g/g BW/24h (6); P < 0.05]. In both controls, AC6−/−, and in AC6loxloxCre mice, dietary Li+ administration increased the water intake (P < 0.05) and reduced the urine osmolality (P < 0.05). Similar to AC6−/− mice, the AC6loxloxCre mice showed under standard conditions lower medullary levels of AQP2 (42% reduction, P < 0.05) and pS256‐AQP2 and (51% reduction, P < 0.05) and the levels were further reduced by respectively 54% and 57% after Li+ administration (P < 0.05). Furthermore, Li+ induced an increase in the numbers of proliferating cell nuclear antigen‐positive cells in the IMCD in both AC6loxloxCre (~2200% increase, P < 0.05) and control mice (~2600% increase, P < 0.05). However, compared to control mice, a greater % of H+‐ATPase B1 subunit‐positive cells were found in the AC6loxloxCre mice under both standard condition (32%, P < 0.05) and after Li+‐administration (33%, P < 0.05). Collectively, AC6 plays a minor role in the development of Li‐NDI, but could have an important function in determining the intercalated‐to‐principal cell ratio, which is altered during Li‐NDI.Support or Funding InformationFunding to R.A.F. is provided by the Novo Nordisk Foundation, the Lundbeck Foundation and Danish Medical Research Council. T.R. is supported by NIDDK 1R01DK110621‐01, the O'Brien Center for Acute Kidney Injury Research Grant P30DK079337, the Diabetes Endocrinology Research Center P30DK063491, the AHA 15BGIA22410018 and Satellite Healthcare (a not‐for‐profit renal care provider).

Functional characterization of lysophosphatidic acid receptor 1 mutants identified in rat cancer tissues

Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts various cellular effects through activation of LPA receptors, LPA1–LPA6, in many types of cells including cancer cells. We recently found several missense mutations of Lpar1 in rat cancer tissues. One of these mutations is located at the extracellular tip of the seventh transmembrane domain of LPA1, and another three mutations are found within the NPXXY motif in the seventh transmembrane domain. These mutants are designated F295S LPA1 and P308S, I310T, and Y311H LPA1, respectively. Here, we examined the functions of these LPA1 mutants. Compared with wild-type (WT) LPA1, F295S, P308S, and I310T LPA1 showed decreased maximal responses in inhibition of cAMP formation, Ca2+ mobilization, and cytoskeletal changes. Y311H LPA1 failed to show LPA-induced cellular responses. However, these LPA1 mutants were internalized in response to LPA exposure. Finally, while WT and F295S LPA1 showed a similar, broad distribution throughout the cell, P308S, I310T, and Y311H LPA1 displayed a restricted cellular distribution and co-localized with the endoplasmic reticulum. These data suggest that the LPA1 mutants perturb LPA signaling in cancer tissues.

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs

We previously demonstrated that the aromatic moiety of Tyr143 within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr143 reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe>Tyr (wild-type)=Trp>Leu>Ala (=0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr143 (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F>i2Y≫i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.

Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca2+ in Nonexcitable Cells.

The β2 adrenergic receptor (β2AR) increases intracellular Ca2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the β2AR promotes Ca2+ mobilization (pEC50 = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca2+ following β2AR stimulation. The increase in cytoplasmic Ca2+ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the β2AR activation. Consistent with this mechanism, β2AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the β2AR-promoted Ca2+ mobilization. Together, these data support a model whereby the β2AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP3-dependent Ca2+ mobilization from intracellular stores. Given that β2AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.

Proterozoic crustal evolution of the Eucla basement, Australia: Implications for destruction of oceanic crust during emergence of Nuna

The crystalline basement beneath the Cretaceous to Cenozoic Bight and Eucla Basins, in Western Australia has received comparatively little attention even though it lies on the eastern margin of one of the most mineral resource endowed regions on the planet. This basement is characterized by a complex geological evolution spanning c. 2billionyears, but paucity of outcrop and younger basin cover present a daunting challenge to understand the basement geology. In this work the composition of the unexposed Proterozoic crystalline basement to the Bight and Eucla Basins is investigated through zircon Hf isotopes and whole rock geochemistry from new drillcore samples. This region includes two geophysically defined basement entities: The Madura Province, containing: 1) c. 1478Ma Sleeper Camp Formation, which has variable isotopic signatures including evolved values interpreted to reflect reworking of rare slivers of hyperextended Archean crust, 2) 1415–1389Ma Haig Cave Supersuite, with mantle-like isotope values interpreted as melting of subduction-modified N-MORB source, and 3) 1181–1125Ma Moodini Supersuite, with juvenile isotopic signatures interpreted to reflect mixed mafic lower-crustal and asthenospheric melts produced at the base of thinned crust. The Coompana Province, to the east of the Madura Province, has three major magmatic components: 1) c. 1610Ma Toolgana Supersuite, with chemical and isotopic characteristics of primitive arc rock, 2) c. 1490Ma Undawidgi Supersuite, with juvenile isotope values consistent with extensional processes involving asthenospheric input and 3) 1192–1140Ma Moodini Supersuite, with strong isotopic similarity to Moodini Supersuite rocks in the Madura Province.This new isotopic and geochemical data shows that the Madura and Coompana regions together represent a huge tract of predominantly juvenile material. Magma sources recognised, include; 1) depleted mantle, producing MORB-like crust at c. 1950Ma, but also contributing to younger magmatism; 2) recycled c. 1950Ma crust reworked in primitive arcs and in intra-plate settings and; 3) minor evolved material representing fragments of hyperextended continent. The observed isotopic evolution pattern is comparable to that of other central Australian Proterozoic provinces, including the Musgrave Province, the northern margin of the Gawler Craton, and components within the Rudall Province. Linking these isotopic signatures defines the Mirning Ocean, and its subducted and underplated equivalents. In a global context we suggest c. 1950Ma crust production reflects the onset of ordered oceanic spreading centres, which swept juvenile crustal fragments into Nuna.

Cyclic AMP Recruits a Discrete Intracellular Ca2+ Store by Unmasking Hypersensitive IP3 Receptors

SummaryInositol 1,4,5-trisphosphate (IP3) stimulates Ca2+ release from the endoplasmic reticulum (ER), and the response is potentiated by 3′,5′-cyclic AMP (cAMP). We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.

Anti-cancer Effects of MW-03, a Novel Indole Compound, by Inducing 15-Hydroxyprostaglandin Dehydrogenase and Cellular Growth Inhibition in the LS174T Human Colon Cancer Cell Line.

Increases in the expression of prostaglandin E2 (PGE2) are widely known to be involved in aberrant growth in the early stage of colon cancer development. We herein demonstrated that the novel indole compound MW-03 reduced PGE2-induced cAMP formation by catalization to an inactive metabolite by inducing 15-hydroxyprostaglandin dehydrogenase through the activation of peroxisome proliferator-activated receptor-γ. MW-03 also inhibited colon cancer cell growth by arresting the cell cycle at the S phase. Although the target of MW-03 for cell cycle inhibition has not yet been identified, these dual anti-cancer effects of MW-03 itself and/or its leading compound(s) on colon cancer cells may reduce colon cancer development and, thus, have potential as a novel treatment for the early stage of this disease.

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure.

Chronic heart failure (HF) is associated with altered signal transduction via β-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of β-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries

Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N- (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid prostacyclin receptor (IP receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP receptor antagonists (CAY10441 and 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]−3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E receptor 3 antagonist enhanced the vasodilation induced by all IP receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP receptor agonists, works as a selective IP receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.

Potent Anti-Platelet Metabolite, 12-HETrE, Inhibits Platelet Activation and Thrombosis In Vivo Via Activation of the IP Receptor

Platelet activation is a critical component of clot formation. Under pathophysiological conditions platelet-mediated clot formation can lead to an occlusive thrombotic event resulting in myocardial infarction and stroke. Regulation of platelet reactivity relies on an understanding of the complex intra-platelet signaling that takes place following activation of various receptors on the platelet surface. Our lab recently identified a novel oxidized metabolite known as 12-HETrE that is formed from 12-lipoxygenase (12-LOX) oxidation of the ω-6 fatty acid DGLA in the platelet. Since DGLA constitutes one of the predominant fatty acids contained in the phospholipids on the platelet membrane and is also a rich source of dietary fatty acid intake through a variety of plant oils, we had originally sought to determine if this metabolite contained inherent regulatory properties on the human platelet. We successfully showed not only that 12-HETrE potently regulates hemostasis and thrombosis in vivo, but additionally identified that the metabolite signals inhibition of platelet activity through a GαS-linked GPCR resulting in formation of cAMP and activation of PKA and VASP. In the current study, we identified for the first time the receptor activated by 12-HETrE in the human platelet. To identify which GPCR is activated by 12-HETrE, we utilized both pharmacologic as well as genetic approaches. Our ex vivo pharmacological data suggested 12-HETrE predominantly signals through the IP receptor in the human platelet. This finding was confirmed using an IP receptor knockout mouse as well as human cell lines expressing or absent IP receptor on their surface. Finally, these findings were confirmed in vivo with wild type or IP knockout mice and assessing the ability of 12-HETrE to inhibit laser-induced thrombosis. Hence, these findings show for the first time that 12-HETrE does in fact regulate inhibition of platelets through the IP receptor. Since 12-HETrE has already been shown to inhibit thrombosis without a significant increase in bleeding diathesis, the current study has elucidated a new target for IP receptor activation and an inhibition of platelet activity and occlusive thrombus formation and should be evaluated for its potential as a new class of anti-thrombotic therapeutics. DisclosuresNo relevant conflicts of interest to declare.

MAGI Proteins Regulate the Trafficking and Signaling of Corticotropin-Releasing Factor Receptor 1 via a Compensatory Mechanism.

Corticotropin-releasing factor (CRF) receptor1 (CRFR1) is associated with psychiatric illness and is a proposed target for the treatment of anxiety and depression. Similar to many G protein-coupled receptors (GPCRs), CRFR1 harbors a PDZ (PSD-95/Disc Large/Zona Occludens)-binding motif at the end of its carboxyl-terminal tail. The interactions of PDZ proteins with GPCRs are crucial for the regulation of receptor function. In the present study, we characterize the interaction of all members of the membrane-associated guanylate kinase with inverted orientation PDZ (MAGI) proteins with CRFR1. We show using co-immunoprecipitation that CRFR1 interacts with MAGI-1 and MAGI-3 in human embryonic kidney (HEK293) cells in a PDZ motif-dependent manner. We find that overexpression as well as knockdown of MAGI proteins result in a significant reduction in CRFR1 endocytosis. This effect is dependent on an intact PDZ binding motif for MAGI-2 and MAGI-3 but not MAGI-1. We show that the alteration in expression levels of MAGI-1, MAGI-2 or MAGI-3 can interfere with β-arrestin recruitment to CRFR1. This could explain the effects observed with receptor internalization. We also find that knockdown of endogenous MAGI-1, MAGI-2 or MAGI-3 in HEK293 cells can lead to an enhancement in ERK1/2 signaling but has no effect on cAMP formation. Interestingly, we observe a compensation effect between MAGI-1 and MAGI-3. Taken together, our data suggest that the MAGI proteins, MAGI-1, MAGI-2 and MAGI-3 can regulate β-arrestin-mediated internalization of CRFR1 as well as its signaling and that there is a compensatory mechanism involved in regulating the function of the MAGI subfamily.

Human DP and EP2 prostanoid receptors take on distinct forms depending on the diverse binding of different ligands.

Human D-type prostanoid (DP) and E-type prostanoid 2 (EP2) receptors are G protein-coupled receptors and are regarded as the most closely related receptors among prostanoid receptors because they are generated by tandem duplication. The DP receptor-cognate ligand, prostaglandin D2 (PGD2 ) has the ability to activate not only DP receptors but also EP2 receptors. Likewise, the EP2 receptor-cognate ligand, prostaglandin E2 (PGE2 ) has the ability to activate DP receptors in addition to EP receptors in order to stimulate cAMP formation. However, since PGD2 and/or PGE2 activate DP and EP2 receptors to similar maximal levels, that is, their similar efficacies, differences between the ligands in each receptor have not yet been determined in detail except for their different affinities. Herein we demonstrated, using an in silico simulation to predict binding patterns among DP or EP2 receptors and PGD2 , PGE2 , or prostaglandin F2α as the reference prostanoid, that DP and EP2 receptors plausibly take on distinct forms depending on the diverse binding of different ligands. Since these ligands have the potential to make these receptors form distinct conformations with discrete signaling pathways, they are consequently regarded as endogenous biased ligands. Moreover, by using functional assays, the susceptibilities of the DP receptors to the noncognate ligands were approximately 10 times lower than those of EP2 receptors. Thus, EP2 receptors seem to be able to distinguish endogenous ligands better than DP receptors, thereby both receptors are plausibly gaining role-sharing functions with respect to one another as the copies of duplicated gene.

Increased sensitivity in the interaction of the dopaminergic/adenosinergic system at the level of the adenylate cyclase activity in the striatum of the “weaver” mouse

The specific antagonistic interaction between dopamine D1 and adenosine A1 receptors (D1/A1), as well as between dopamine D2 and adenosine A2a receptors (D2/A2a) exist not only at the receptor/receptor level, but also at the level of the secondary messengers. In this study, we examined the possible changes in these interactions at the level of cAMP formation in membrane preparation from “weaver” mouse striatum (a genetic model of Parkinson disease), by using specific agonists of these receptors. We also examined in the striatum of the “weaver” mouse the interaction between D1 and D2 dopamine receptors.Our results showed that in the striatum of “weaver” mice: a) the cAMP synthesis induced by D1 receptor activation (SKF 38393), was significantly reduced compared to control mice, while A1 receptor activation (L-PIA) leaded to a more intense inhibition of the D1-induced cAMP-formation compared to the controls, b) the cAMP synthesis which was induced by A2a receptor activation (CGS 21680), was significantly increased compared to the control mice. The specific D2 receptor agonist Quinpirole, added in low concentrations, caused a significant reduction of the A2a-induced cAMP formation, which was not observed in the control mouse. Furthermore, the D1 receptor induced cAMP synthesis was significantly higher in control compared to “weaver” striatum, which was more efficiently downregulated by D2 receptor agonist Quinpirole.These results suggest that the sensitivity to D1 and A2a receptor agonists is altered and that the interaction between D1/A1 and D2/A2a receptors is enhanced in the striatum of the “weaver” mutation, while an uncoupling between D1 and D2 receptors was observed. Since the adenylate cyclase basal activity did not differ between “weaver” and control striatum, the above-mentioned changes seem to be due to alterations in the function of the adenosine/dopamine receptors and their coupling to the G-proteins.

EP 12. Fingolimod promotes nerve regeneration by modulating Lysophospholipid signaling

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are pleiotropic signaling molecules with a broad range of physiological functions. Targeting the S1P 1 receptor on lymphocytes with the immunomodulatory drug fingolimod has proven effective in the treatment of multiple sclerosis. An emerging body of experimental evidence points to additional direct effects on cells of the central and peripheral nervous system. Furthermore, fingolimod has been reported to reduce LPA synthesis via inhibition of the lysophospholipase autotaxin. Here we investigated whether modulation of particular signaling aspects of S1P as well as LPA by fingolimod might propagate peripheral nerve regeneration in vivo and independent of its anti-inflammatory potency. To distinguish potential direct nerve specific effects from the established immunosuppressive action of fingolimod, we performed sciatic nerve crush in wildtype C57BL/6 as well as in immunodeficient Rag 1 - / - and Foxn 1 - / - mice. Analyses were based on walking track analysis and electrophysiology, histology, cAMP formation as well as oxidative stress assessment by dinitrophenyl-labeling of free protein carbonyls. Quantification of different LPA species was performed by liquid chromatography coupled to tandem mass spectrometry. Furthermore, functional consequences of LPA level reduction by the autotaxin inhibitor PF-8380 were investigated. Clinical, electrophysiological and histological measures indicated an improvement of nerve regeneration under fingolimod treatment that is partly independent of its anti-inflammatory properties. Fingolimod induced an elevation of axonal cAMP, a crucial factor for axonal outgrowth. Consistently, immunohistochemistry indicated S1P 1 receptor internalization in cells of the sciatic nerve. Additionally, fingolimod attenuated oxidative tissue damage and significantly reduced LPA levels in the injured nerve. PF-8380 induced reduction of LPA levels correlated with improved myelin thickness. Collectively, our work demonstrates that modulation of lysophospholipid signaling in the peripheral nervous system by fingolimod may enhance nerve regeneration by acting on multiple molecular and cellular levels and partly independent of its anti-inflammatory effects. In addition to the established role of fingolimod as a modulator of S1P receptor mediated signaling, the inhibition of LPA synthesis may represent a yet unrecognized mechanism that contributes to the presumptive remyelinating effect of fingolimod.

Structure-guided development of heterodimer-selective GPCR ligands.

Crystal structures of G protein-coupled receptor (GPCR) ligand complexes allow a rational design of novel molecular probes and drugs. Here we report the structure-guided design, chemical synthesis and biological investigations of bivalent ligands for dopamine D2 receptor/neurotensin NTS1 receptor (D2R/NTS1R) heterodimers. The compounds of types 1–3 consist of three different D2R pharmacophores bound to an affinity-generating lipophilic appendage, a polyethylene glycol-based linker and the NTS1R agonist NT(8-13). The bivalent ligands show binding affinity in the picomolar range for cells coexpressing both GPCRs and unprecedented selectivity (up to three orders of magnitude), compared with cells that only express D2Rs. A functional switch is observed for the bivalent ligands 3b,c inhibiting cAMP formation in cells singly expressing D2Rs but stimulating cAMP accumulation in D2R/NTS1R-coexpressing cells. Moreover, the newly synthesized bivalent ligands show a strong, predominantly NTS1R-mediated β-arrestin-2 recruitment at the D2R/NTS1R-coexpressing cells.