Luciferase-based GloSensor™ cAMP assay: Temperature optimization and application to cell-based kinetic studies.

G protein-coupled receptors (GPCRs) are an important receptor superfamily and common therapeutic targets. The second messenger cyclic adenosine monophosphate (cAMP) is a key mediator in many GPCR signaling pathways. Monitoring intracellular cAMP levels can help identify orthosteric agonists and antagonists, as well as allosteric modulators. In this regard, luminescence-based biosensors have revolutionized our ability to monitor GPCR signaling kinetics. The GloSensor™ cAMP assay enables real-time monitoring of signaling downstream of many GPCRs. However, it is crucial to optimize assay conditions such as temperature. As well, it has not been reported whether the effects of temperature on biosensor activity are reversible. Here, we describe the temperature sensitivity and reversibility of the GloSensor™ cAMP assay, and which GloSensor™ version is optimal for measuring cytosolic cAMP. We also present a detailed protocol for monitoring cAMP levels in live cells expressing endogenous or exogenous GPCRs. Temperature optimization studies were carried out using HEK293H cells transiently transfected with the adenosine receptor A2a and the GloSensor™ plasmid (pGloSensor-20F or -22F). We found that preincubation and luminescence reading at room temperature were optimal as compared to higher temperatures. As well, the GloSensor-22F biosensor had a superior signal-to-background ratio and the effect of temperature on biosensor activity was reversible. However, thermal instability of the biosensor may pose a problem for in vivo studies. Nevertheless, the GloSensor™ cAMP assay can be applied to analyze signaling by a wide range of GPCRs for drug discovery purposes.

Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4).

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

Maternal diabetes up-regulates NOX2 and enhances myocardial ischaemia/reperfusion injury in adult offspring.

Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up-regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose-tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF-Akt (protein kinase B)-mammalian target of rapamycin (mTOR)-NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose-induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF-Akt-mTOR-NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.

Regulator of G-protein signaling 2 (RGS2) suppresses premature calcium release in mouse eggs.

During oocyte maturation, capacity and sensitivity of Ca(2+) signaling machinery increases dramatically, preparing the metaphase II (MII)-arrested egg for fertilization. Upon sperm-egg fusion, Ca(2+) release from IP3-sensitive endoplasmic reticulum stores results in cytoplasmic Ca(2+) oscillations that drive egg activation and initiate early embryo development. Premature Ca(2+) release can cause parthenogenetic activation prior to fertilization; thus, preventing inappropriate Ca(2+) signaling is crucial for ensuring robust MII arrest. Here, we show that regulator of G-protein signaling 2 (RGS2) suppresses Ca(2+) release in MII eggs. Rgs2 mRNA was recruited for translation during oocyte maturation, resulting in ∼ 20-fold more RGS2 protein in MII eggs than in fully grown immature oocytes. Rgs2-siRNA-injected oocytes matured to MII; however, they had increased sensitivity to low pH and acetylcholine (ACh), which caused inappropriate Ca(2+) release and premature egg activation. When matured in vitro, RGS2-depleted eggs underwent spontaneous Ca(2+) increases that were sufficient to cause premature zona pellucida conversion. Rgs2(-/-) females had reduced litter sizes, and their eggs had increased sensitivity to low pH and ACh. Rgs2(-/-) eggs also underwent premature zona pellucida conversion in vivo. These findings indicate that RGS2 functions as a brake to suppress premature Ca(2+) release in eggs that are poised on the brink of development.

Cardiomyocyte specific overexpression of a 37 amino acid domain of regulator of G protein signalling 2 inhibits cardiac hypertrophy and improves function in response to pressure overload in mice.

Regulator of G protein signalling 2 (RGS2) is known to play a protective role in maladaptive cardiac hypertrophy and heart failure via its ability to inhibit Gq- and Gs- mediated GPCR signalling. We previously demonstrated that RGS2 can also inhibit protein translation and can thereby attenuate cell growth. This G protein-independent inhibitory effect has been mapped to a 37 amino acid domain (RGS2eb) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed in neonatal rat cardiomyocytes, RGS2eb attenuates both protein synthesis and hypertrophy induced by Gq- and Gs- activating agents. In the current study, we investigated the potential cardioprotective role of RGS2eb by determining whether RGS2eb transgenic (RGS2eb TG) mice with cardiomyocyte specific overexpression of RGS2eb show resistance to the development of hypertrophy in comparison to wild-type (WT) controls. Using transverse aortic constriction (TAC) in a pressure-overload hypertrophy model, we demonstrated that cardiac hypertrophy was inhibited in RGS2eb TG mice compared to WT controls following four weeks of TAC. Expression of the hypertrophic markers atrial natriuretic peptide (ANP) and ß-myosin heavy chain (MHC-ß) was also reduced in RGS2eb TG compared to WT TAC animals. Furthermore, cardiac function in RGS2eb TG TAC mice was significantly improved compared to WT TAC mice. Notably, cardiomyocyte cell size was significantly decreased in TG compared to WT TAC mice. These results suggest that RGS2 may limit pathological cardiac hypertrophy at least in part via the function of its eIF2B-binding domain.

RGS proteins destroy spare receptors: Effects of GPCR-interacting proteins and signal deamplification on measurements of GPCR agonist potency.

Many GPCRs are able to activate multiple distinct signaling pathways, and these may include biochemical cascades activated via either heterotrimeric G proteins or by ß-arrestins. The relative potencies and/or efficacies among a series of agonists that act on a common receptor can vary depending upon which signaling pathway is being activated. This phenomenon is known as biased signaling or functional selectivity, and is presumed to reflect underlying differences in ligand binding affinities for alternate conformational states of the receptor. The first part of this review discusses how various cellular GPCR interacting proteins (GIPs) can influence receptor conformation and thereby affect ligand-receptor interactions and contribute to signaling bias. Upon activation, receptors trigger biochemical cascades that lead to altered cellular function, and measuring points along the cascade (e.g., second messenger production) conveys information about receptor activity. As a signal continues along its way, the observed concentration dependence of a GPCR ligand may change due to amplification and saturation of biochemical steps. The second part of this review considers additional cellular factors that affect signal processing, focusing mainly on structural elements and deamplification mechanisms, and discusses the relevance of these to measurements of potency and functional selectivity.

GPCR Retreat 2014: a good view leads to many discoveries!

The GPCR gods smiled on us last year as the 15th Annual GPCR Retreat was held last October 2nd-4th in Bromont, Québec. The fall colors were at their peak and the meeting attendees were also in fine form. The program was one of the best we have seen at any GPCR-related meeting in years and there was a great deal of excitement about new methodological approaches to understanding receptor biology, new concepts in GPCR signaling and a continued emphasis on translation of these discoveries. This year was also the first year we opened the meeting with a short course on biased agonism and how to measure and analyze it.

Regulation of RGS5 GAP activity by GPSM3.

Heterotrimeric G protein signaling is limited by intracellular proteins that impede the binding of or accelerate the hydrolysis of the activating nucleotide GTP, exemplified respectively by the G protein-signaling modifier (GPSM) and regulator of G protein-signaling (RGS) families of proteins. Little is known about how members of these groups of proteins might influence the impact of the other on G protein activity. In the present study, we have identified novel binding and functional interactions between GPSM3 (also known as activator of G protein-signaling 4 (AGS4) or G18) and RGS5, both of which were found to be expressed in primary rat aortic smooth muscle cell cultures. The binding of GPSM3 to RGS5 appears to be selective as no interactions were detected with other RGS proteins tested. In solution-based experiments, the addition of GPSM3 was found to enhance the ability of RGS5 to accelerate GTP hydrolysis by Gαi1 but not that of RGS4. In membrane-based assays utilizing M2 muscarinic receptor-activated Gαi1, GPSM3 decreased the rate of GTP hydrolysis in the presence of RGS4 but not RGS5, suggesting that the enhancement of RGS5 activity by GPSM3 is maintained under these conditions and/or that the binding of RGS5 to GPSM3 impedes its inhibitory effect on GTP turnover. Overall these findings show that it is possible for GPSM and RGS proteins to bind to one another to produce distinct regulatory effects on heterotrimeric G protein activity.

The Ras-binding domain region of RGS14 regulates its functional interactions with heterotrimeric G proteins.

RGS14 is a 60 kDa protein that contains a regulator of G protein signaling (RGS) domain near its N-terminus, a central region containing a pair of tandem Ras-binding domains (RBD), and a GPSM (G protein signaling modulator) domain (a.k.a. Gi/o-Loco binding [GoLoco] motif) near its C-terminus. The RGS domain of RGS14 exhibits GTPase accelerating protein (GAP) activity toward Gαi/o proteins, while its GPSM domain acts as a guanine nucleotide dissociation inhibitor (GDI) on Gαi1 and Gαi3. In the current study, we investigate the contribution of different domains of RGS14 to its biochemical functions. Here we show that the full-length protein has a greater GTPase activating activity but a weaker inhibition of nucleotide dissociation relative to its isolated RGS and GPSM regions, respectively. Our data suggest that these differences may be attributable to an inter-domain interaction within RGS14 that promotes the activity of the RGS domain, but simultaneously inhibits the activity of the GPSM domain. The RBD region seems to play an essential role in this regulatory activity. Moreover, this region of RGS14 is also able to bind to members of the B/R4 subfamily of RGS proteins and enhance their effects on GPCR-activated Gi/o proteins. Overall, our results suggest a mechanism wherein the RBD region associates with the RGS domain region, producing an intramolecular interaction within RGS14 that enhances the GTPase activating function of its RGS domain while disfavoring the negative effect of its GPSM domain on nucleotide dissociation.

GPCR Retreat 2012: timing is everything.

In London, Ontario, the 13th Annual Joint meeting of the Great Lakes GPCR Retreat and the Club des Récepteurs à Sept Domaines Transmembranaires (known simply as the GPCR Retreat) was held on 17-19 October 2012, organized by Steve Ferguson and Peter Chidiac. This meeting gathered together a core group of investigators from Michigan, Ontario and Québec and has steadily increased its attendance in both the eastern (Europe) and western (USA, Canada) directions. This year's buzz naturally centered around the Nobel Prize in Chemistry, which was won the week before by Brian Kobilka and Robert Lefkowitz for their work on receptor structure and function. Michel Bouvier provided a heartfelt tribute to one of the attendees, Marc Caron, a pioneer in the GPCR field, has made many contributions to the work that led to this year's Nobel Prize. The meeting featured interesting sessions on the physiological roles of GPCRs in the nervous system, circadian biology and cancer, dealing at the cellular and molecular level with GPCR, G protein and effector structure and function, regulation and trafficking--with an overall focus on how to move molecular pharmacology in vivo.

RGS2 is a component of the cellular stress response.

Regulator of G protein signaling (RGS) proteins are GTPase accelerating proteins for heterotrimeric G protein α-subunits. RGS2 has recently been shown to have additional G protein-independent functions including control of ion channel currents, microtubule polymerization, and protein synthesis. Cellular levels of RGS2 mRNA and protein are upregulated in response to various forms of stress suggesting that it may be a stress-adaptive protein; however, direct evidence to support this notion has remained elusive. In this report, we show that thermal stress upregulates RGS2 expression and this serves to arrest de novo protein synthesis. The latter is an established cellular response to stress. Inhibiting the stress-induced RGS2 upregulation by way of siRNA knockdown diminished the repression of global protein synthesis. The collective results of our study implicate RGS2 upregulation as a cellular mechanism of controlling de novo protein synthesis in response to stress. This work provides greater insight into the stress proteome and the role of RGS2.

Inhibition of Na/K-ATPase promotes myocardial tumor necrosis factor-alpha protein expression and cardiac dysfunction via calcium/mTOR signaling in endotoxemia.

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that causes cardiac dysfunction during sepsis. Na/K-ATPase regulates intracellular Ca(2+), which activates mammalian target of rapamycin (mTOR), a regulator of protein synthesis. The aim of this study was to investigate the role of Na/K-ATPase/mTOR signaling in myocardial TNF-α expression during endotoxemia. Results showed that treatment with LPS decreased Na/K-ATPase activity in the myocardium in vivo and in cultured neonatal cardiomyocytes. Inhibition of Na/K-ATPase by ouabain enhanced LPS-induced myocardial TNF-α protein production, but had no effect on TNF-α mRNA expression. More importantly, ouabain further decreased in vivo cardiac function in endotoxemic mice, which was blocked by etanercept, a TNF-α antagonist. LPS-induced reduction in Na/K-ATPase activity was prevented by inhibition of PI3K, Rac1 and NADPH oxidase using LY294002, a dominant-negative Rac1 adenovirus (Ad-Rac1N17) and apocynin, respectively. To assess the role of Rac1 in Ca(2+) handling, Ca(2+) transients in adult cardiomyocytes from cardiomyocyte-specific Rac1 knockout (Rac1(CKO)) and wild-type (WT) mice were determined. LPS increased intracellular Ca(2+) in WT but not in Rac1(CKO) cardiomyocytes. Furthermore, LPS rapidly increased mTOR phosphorylation in cardiomyocytes, which was blocked by Rac1N17 and an inhibitor of calmodulin-dependent protein kinases (CaMKs) KN93, but enhanced by ouabain. Rapamycin, an inhibitor of mTOR suppressed TNF-α protein levels without any significant effect on its mRNA expression or global protein synthesis. In conclusion, myocardial Na/K-ATPase activity is inhibited during endotoxemia via PI3K/Rac1/NADPH oxidase activation. Inhibition of Na/K-ATPase activates Ca(2+)/CaMK/mTOR signaling, which promotes myocardial TNF-α protein production and cardiac dysfunction during endotoxemia.

The proline-rich N-terminal domain of G18 exhibits a novel G protein regulatory function.

The protein G18 (also known as AGS4 or GPSM3) contains three conserved GoLoco/GPR domains in its central and C-terminal regions that bind to inactive Galpha(i), whereas the N-terminal region has not been previously characterized. We investigated whether this domain might itself regulate G protein activity by assessing the abilities of G18 and mutants thereof to modulate the nucleotide binding and hydrolytic properties of Galpha(i1) and Galpha(o). Surprisingly, in the presence of fluoroaluminate (AlF(4)(-)) both G proteins bound strongly to full-length G18 (G18wt) and to its isolated N-terminal domain (G18DeltaC) but not to its GoLoco region (DeltaNG18). Thus, it appears that its N-terminal domain promotes G18 binding to fluoroaluminate-activated Galpha(i/o). Neither G18wt nor any G18 mutant affected the GTPase activity of Galpha(i1) or Galpha(o). In contrast, complex effects were noted with respect to nucleotide binding. As inferred by the binding of [(35)S]GTPgammaS (guanosine 5'-O-[gamma-thio]triphosphate) to Galpha(i1), the isolated GoLoco region as expected acted as a guanine nucleotide dissociation inhibitor, whereas the N-terminal region exhibited a previously unknown guanine nucleotide exchange factor effect on this G protein. On the other hand, the N terminus inhibited [(35)S]GTPgammaS binding to Galpha(o), albeit to a lesser extent than the effect of the GoLoco region on Galpha(i1). Taken together, our results identify the N-terminal region of G18 as a novel G protein-interacting domain that may have distinct regulatory effects within the G(i/o) subfamily, and thus, it could potentially play a role in differentiating signals between these related G proteins.

Cardiomyocyte-specific overexpression of human stem cell factor improves cardiac function and survival after myocardial infarction in mice.

BACKGROUND: Soluble stem cell factor (SCF) has been shown to mobilize bone marrow stem cells and improve cardiac repair after myocardial infarction (MI). However, the effect of membrane-associated SCF on cardiac remodeling after MI is not known. The present study investigated the effects of cardiomyocyte-specific overexpression of the membrane-associated isoform of human SCF (hSCF) on cardiac function after MI. METHODS AND RESULTS: A novel mouse model with tetracycline-inducible and cardiac-specific overexpression of membrane-associated hSCF was generated. MI was induced by left coronary artery ligation. Thirty-day mortality after MI was decreased in hSCF/tetracycline transactivator (tTA) compared with wild-type mice. In vivo cardiac function was significantly improved in hSCF/tTA mice at 5 and 30 days after MI compared with wild-type mice. Endothelial progenitor cell recruitment and capillary density were increased and myocardial apoptosis was decreased in the peri-infarct area of hSCF/tTA mice. Myocyte size was decreased in hSCF/tTA mice 30 days after MI compared with WT mice. Furthermore, hSCF overexpression promoted de novo angiogenesis as assessed by matrigel implantation into the left ventricular myocardium. CONCLUSIONS: Cardiomyocyte-specific overexpression of hSCF improves myocardial function and survival after MI. These beneficial effects of hSCF may result from increases in endothelial progenitor cell recruitment and neovascularization and decreases in myocardial apoptosis and cardiac remodeling.

Evidence for enhanced M3 muscarinic receptor function and sensitivity to atrial arrhythmia in the RGS2-deficient mouse.

Atrial fibrillation (AF) is the most common arrhythmia seen in general practice. Muscarinic ACh receptors (M2R, M3R) are involved in vagally induced AF. M2R and M3R activate the heterotrimeric G proteins, G(i) and G(q), respectively, by promoting GTP binding, and these in turn activate distinct K(+) channels. Signaling is terminated by GTP hydrolysis, a process accelerated by regulator of G protein signaling (RGS) proteins. RGS2 is selective for G(q) and thus may regulate atrial M3R signaling. We hypothesized that knockout of RGS2 (RGS2(-/-)) would render the atria more susceptible to electrically induced AF. One-month-old male RGS2(-/-) and C57BL/6 wild-type (WT) mice were instrumented for intracardiac electrophysiology. Atrial effective refractory periods (AERPs) were also determined in the absence and presence of carbachol, atropine, and/or the selective M3R antagonist darifenacin. Susceptibility to electrically induced AF used burst pacing and programmed electrical stimulation with one extrastimulus. Real-time RT-PCR measured atrial and ventricular content of RGS2, RGS4, M2R, M3R, and M4R mRNA. AERP was lower in RGS2(-/-) compared with WT mice in both the high right atrium (HRA) (30 +/- 1 vs. 34 +/- 1 ms, P < 0.05) and mid right atrium (MRA) (21 +/- 1 vs. 24 +/- 1 ms, P < 0.05). Darifenacin eliminated this difference (HRA: 37 +/- 2 vs. 39 +/- 2 ms, and MRA: 30 +/- 2 vs. 30 +/- 1, P > 0.4). RGS2(-/-) were more susceptible than WT mice to atrial tachycardia/fibrillation (AT/F) induction (11/22 vs. 1/25, respectively, P < 0.05). Muscarinic receptor expression did not differ between strains, whereas M2R expression was 70-fold higher than M3R (P < 0.01). These results suggest that RGS2 is an important cholinergic regulator in the atrium and that RGS2(-/-) mice have enhanced susceptibility to AT/F via enhanced M3 muscarinic receptor activity.

Hippocampal long-term potentiation is enhanced in urethane-anesthetized RGS2 knockout mice.

RGS2 is a member of the regulator of G-protein signaling (RGS) family and has been implicated in cellular mechanisms associated with neuronal plasticity. Long-term potentiation (LTP) of RGS2 knockout and wild-type mice was examined at the Schaffer collaterals to CA1 pathway in urethane-anesthetized mice in vivo to examine RGS2's possible role in the regulation of potentiation. As compared to wild-type mice, RGS2 knockouts demonstrated much stronger LTP of the extracellular population spikes at the somatic and dendritic layers in CA1 region and more pronounced LTP of the population excitatory postsynaptic current sink. Under baseline conditions, RGS2 knockouts showed lower paired-pulse facilitation of the excitatory postsynaptic potentials and associated current sinks in vivo as compared with wild-type mice. The data show for the first time that RGS2 deficient mice in vivo differ from wild-type mice in both short-term and long-term synaptic plasticity suggesting that RGS2 serves as a negative regulator of long-term synaptic plasticity.

RGS2 promotes the translation of stress-associated proteins ATF4 and CHOP via its eIF2B-inhibitory domain.

Regulator of G protein signaling 2 (RGS2) is upregulated by multiple forms of stress and can augment translational attenuation associated with the phosphorylation of the initiation factor eIF2, a hallmark of several stress-induced coping mechanisms. Under stress-induced translational inhibition, key factors, such as ATF4, are selectively expressed via alternative translation mechanisms. These factors are known to regulate molecular switches that control cell fate by regulating pro-survival and pro-apoptotic signals. The molecular mechanisms that balance these opposing responses to stresses are unclear. The present results suggest that RGS2 may be an important regulatory component in the cellular stress response through its translational control abilities. Previously, we have shown that RGS2 can interact with the translation initiation factor, eIF2B, and inhibit de novo protein synthesis. Here, we demonstrate that the expression of either full length RGS2 or its eIF2B-interacting domain (RGS2eb) significantly increases levels of ATF4 and CHOP, both of which are linked to stress-induced apoptosis. Furthermore, we show that these effects are translationally regulated and independent of eIF2 phosphorylation. The present results thus point to a novel function of RGS2 in the stress response directly related to its ability to reduce global protein synthesis.

Toward Defining the Pharmacophore for Positive Allosteric Modulation of PTH1 Receptor Signaling by Extracellular Nucleotides.

The parathyroid hormone 1 receptor (PTH1R) is a Class B G-protein-coupled receptor that is a target for osteoporosis therapeutics. Activated PTH1R couples through Gs to the stimulation of adenylyl cyclase. As well, ß-arrestin is recruited to PTH1R leading to receptor internalization and MAPK/ERK signaling. Previously, we reported that the agonist potency of PTH1R is increased in the presence of extracellular ATP, which acts as a positive allosteric modulator of PTH signaling. Another nucleotide, cytidine 5'-monophosphate (CMP), also enhances PTH1R signaling, suggesting that ATP and CMP share a moiety responsible for positive allostery, possibly ribose-5-phosphate. Therefore, we examined the effect of extracellular sugar phosphates on PTH1R signaling. cAMP levels and ß-arrestin recruitment were monitored using luminescence-based assays. Alone, ribose-5-phosphate had no detectable effect on adenylyl cyclase activity in UMR-106 rat osteoblastic cells, which endogenously express PTH1R. However, ribose-5-phosphate markedly enhanced the activation of adenylyl cyclase induced by PTH. Other sugar phosphates, including glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-bisphosphate, also potentiated PTH-induced adenylyl cyclase activation. As well, some sugar phosphates enhanced PTH-induced ß-arrestin recruitment to human PTH1R heterologously expressed in HEK293H cells. Interestingly, the effects of glucose-1-phosphate were greater than those of its isomer glucose-6-phosphate. Our results suggest that phosphorylated monosaccharides such as ribose-5-phosphate contain the pharmacophore for positive allosteric modulation of PTH1R. At least in some cases, the extent of modulation depends on the position of the phosphate group. Knowledge of the pharmacophore may permit future development of positive allosteric modulators to increase the therapeutic efficacy of PTH1R agonists.

Regulators of G protein signalling: a spotlight on emerging functions in the cardiovascular system.

Regulator of G protein signalling (RGS) proteins are GTPase-activating proteins for heterotrimeric G protein alpha subunits, and are therefore physiologically and pathophysiologically important negative regulators of G-protein-coupled receptor signalling in the cardiovascular system. Owing to the functional redundancy of many of the 20 RGS, and more than 20 RGS-like, proteins even within a single cell, animal models shedding light on the functions of individual RGS proteins are often missing. Nevertheless, RGS2 is a member of this protein family, for which specific functions in the vasculature and the heart are now emerging. Recent data show that the 519-amino acid RGS3, the only RGS protein with an additional G protein betagamma dimer binding domain, largely alters the signalling of G(i) proteins to the monomeric GTPases Rac1 and RhoA in cardiomyocytes. In addition, an alternative approach using transgenic animals expressing RGS-resistant G protein alpha subunits now highlights the contributions of RGS proteins to distinct signalling pathways in the heart.

Extracellular nucleotides enhance agonist potency at the parathyroid hormone 1 receptor.

Parathyroid hormone (PTH) activates the PTH/PTH-related peptide receptor (PTH1R) on osteoblasts and other target cells. Mechanical stimulation of cells, including osteoblasts, causes release of nucleotides such as ATP into the extracellular fluid. In addition to its role as an energy source, ATP serves as an agonist at P2 receptors and an allosteric regulator of many proteins. We investigated the effects of concentrations of extracellular ATP, comparable to those that activate low affinity P2X7 receptors, on PTH1R signaling. Cyclic AMP levels were monitored in real-time using a bioluminescence reporter and ß-arrestin recruitment to PTH1R was followed using a complementation-based luminescence assay. ATP markedly enhanced cyclic AMP and ß-arrestin signaling as well as downstream activation of CREB. CMP - a nucleotide that lacks a high energy bond and does not activate P2 receptors - mimicked this effect of ATP. Moreover, potentiation was not inhibited by P2 receptor antagonists, including a specific blocker of P2X7. Thus, nucleotide-induced potentiation of signaling pathways was independent of P2 receptor signaling. ATP and CMP reduced the concentration of PTH (1-34) required to produce a half-maximal cyclic AMP or ß-arrestin response, with no evident change in maximal receptor activity. Increased potency was similarly apparent with PTH1R agonists PTH (1-14) and PTH-related peptide (1-34). These observations suggest that extracellular nucleotides increase agonist affinity, efficacy or both, and are consistent with modulation of signaling at the level of the receptor or a closely associated protein. Taken together, our findings establish that ATP enhances PTH1R signaling through a heretofore unrecognized allosteric mechanism.