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Agonists are defined as the ligands that activate intracellular signaling and evoke cellular responses. Synthetic and endogenous agonists should bind specific amino acids to activate G protein-coupled receptor (GPCR). Agonists that induce maximal responses are full agonists. Partial agonists cannot induce full responses unlike full agonists. In definition, antagonists inhibit agonist-stimulated responses by binding to orthosteric or allosteric sites. Antagonists modulate agonist-induced responses and are often related with inverse agonist activity. However, the relationship between antagonists and partial agonists is complex. An antagonist behaves as a partial agonist when the constitutive activity of the GPCR is high. In contrast, a partial agonist with very weak intrinsic activity may be classified as an antagonist. Thus, antagonisms of the compounds are influenced by constitutive activity of GPCRs, intrinsic activity and differences in the binding sites of GPCRs. Since "antagonism" has been revealed to have multiple aspects and more complex than previously thought, it may be difficult to classify each compound as simply "agonist" or "antagonist" as before. In this review, we discuss the recent findings and perspectives on the pharmacology of GPCR-binding antagonists, inverse agonists, and signaling.

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Nonalcoholic fatty liver disease (NAFLD) arises from mitochondrial dysfunction under sustained imbalance between energy intake and expenditure, but the underlying mechanisms controlling mitochondrial respiration have not been entirely understood. Heterotrimeric G proteins converge with activated GPCRs to modulate cell-signaling pathways to maintain metabolic homeostasis. Here, we investigated the regulatory role of G protein α12 (Gα12) on hepatic lipid metabolism and whole-body energy expenditure in mice. Fasting increased Gα12 levels in mouse liver. Gα12 ablation markedly augmented fasting-induced hepatic fat accumulation. cDNA microarray analysis from Gna12-KO liver revealed that the Gα12-signaling pathway regulated sirtuin 1 (SIRT1) and PPARα, which are responsible for mitochondrial respiration. Defective induction of SIRT1 upon fasting was observed in the liver of Gna12-KO mice, which was reversed by lentivirus-mediated Gα12 overexpression in hepatocytes. Mechanistically, Gα12 stabilized SIRT1 protein through transcriptional induction of ubiquitin-specific peptidase 22 (USP22) via HIF-1α increase. Gα12 levels were markedly diminished in liver biopsies from NAFLD patients. Consistently, Gna12-KO mice fed a high-fat diet displayed greater susceptibility to diet-induced liver steatosis and obesity due to decrease in energy expenditure. Our results demonstrate that Gα12 regulates SIRT1-dependent mitochondrial respiration through HIF-1α-dependent USP22 induction, identifying Gα12 as an upstream molecule that contributes to the regulation of mitochondrial energy expenditure.

Erk5 belongs to the mitogen-activated protein kinase (MAPK) family. Following its phosphorylation by Mek5, Erk5 modulates several signaling pathways in a number of cell types. In this study, we demonstrated that Erk5 inactivation in mesenchymal cells causes abnormalities in skeletal development by inducing Sox9, an important transcription factor of skeletogenesis. We further demonstrate that Erk5 directly phosphorylates and activates Smurf2 (a ubiquitin E3 ligase) at Thr 249 , which promotes the proteasomal degradation of Smad proteins and phosphorylates Smad1 at Ser 206 in the linker region known to trigger its proteasomal degradation by Smurf1. Smads transcriptionally activated the expression of Sox9 in mesenchymal cells. Accordingly, removal of one Sox9 allele in mesenchymal cells from Erk5-deficient mice rescued some abnormalities of skeletogenesis. These findings highlight the importance of the Mek5-Erk5-Smurf-Smad-Sox9 axis in mammalian skeletogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called "biased ligands" elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β(1)-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β(1)-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β(1)-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers.

An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS(-)) regulates the metabolism and signaling actions of various electrophiles. HS(-) reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS(-) biosynthesis. Cardiac HS(-), in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS(-) on myocardial infarction-associated heart failure. Thus, this study reveals HS(-)-induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling.

The overexpression of mitochondrial transcription factor A (TFAM) attenuates the decrease in mtDNA copy number after myocardial infarction, ameliorates pathological hypertrophy, and markedly improves survival. However, non-transgenic strategy to increase mtDNA for the treatment of pathological hypertrophy remains unknown. We produced recombinant human TFAM protein (rhTFAM). rhTFAM rapidly entered into mitochondria of cultured cardiac myocytes. rhTFAM increased mtDNA and abolished the activation of nuclear factor of activated T cells (NFAT), which is well known to activate pathological hypertrophy. rhTFAM attenuated subsequent morphological hypertrophy of myocytes as well. rhTFAM would be an attractive molecule in attenuating cardiac pathological hypertrophy., (Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

β-Arrestins (β-arrestin-1 and β-arrestin-2) were first identified as proteins that have the ability to desensitize G protein-coupled receptors (GPCRs). However, it has recently been found that β-arrestins can activate signaling pathways independent of G protein activation. The diversity of these signaling pathways has also been recognized. This leads to an appreciation of β-arrestin-biased agonists, which is a new class of drugs that selectively activate β-arrestin-mediated signaling without G protein activation. In this review, we will discuss the recent advance of β-arrestin-mediated signaling pathways, including a brief account of different biased agonists, their pharmacological applications, and novel β-arrestin research.

Angiotensin II (Ang II) is a major vasoactive peptide of the renin-angiotensin system. Ang II is originally found as one of potent vasoconstrictors, but is now attracted attention as an essential mediator of many cardiovascular problems, including endothelial dysfunction, arrhythmia and structural remodeling of cardiovascular systems. Most of the known pathophysiological effects of Ang II are mediated through Ang type1 receptors (AT(1)Rs), and the up-regulation of AT(1)Rs is one of important causes by which Ang II can contribute to cardiovascular diseases. A growing body of evidence has suggested that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in the regulation of AT(1)R signaling. In cardiac fibroblasts, stimulation with cytokines or bacterial toxins induces AT(1)R up-regulation through NADPH oxidase-dependent ROS production. In contrast, nitric oxide (NO) decreases AT(1)R density through cysteine modification (S-nitrosylation) of a transcriptional factor, nuclear factor κB (NF-κB). The difference between the effects of ROS and NO on AT(1)R expression may be caused by the difference between intracellular location of ROS signaling and that of NO signaling, as the agonist-induced S-nitrosylation of NF-κB requires a local interaction between NO synthase (NOS) and NF-κB in the perinuclear region. Thus, the spatial and temporal regulation of cysteine modification by ROS or RNS may underlie the resultant changes of AT(1)R signaling induced by agonist stimulation., (Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.)

Rationale: Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized., Objective: We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca(2+) channel known to positively regulate prohypertrophic calcineurin-nuclear factor of activated T cells (NFAT) signaling., Methods and Results: In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca(2+) influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II-induced cardiac hypertrophy in mice., Conclusions: Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling.

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)-beta. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Galpha(12/13)) in cardiomyocytes suppressed pressure overload-induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF-beta, connective tissue growth factor, and periostin) and Ang-converting enzyme (ACE) was suppressed by the functional inhibition of Galpha(12/13). The expression of these fibrogenic genes through Galpha(12/13) by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G-protein-coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Galpha(12/13) in cardiomyocytes by the extracellular nucleotides-stimulated P2Y(6) receptor triggers fibrosis in pressure overload-induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF-beta.

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF-dependent neuritogenesis enhancer potential of a food-derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor-initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic-promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6-methylsulfinylhexyl isothiocyanate (6-HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6-HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low-concentration of NGF that alone was insufficient to induce neuronal differentiation. 6-HITC also facilitated the sustained-phosphorylation of the extracellular signal-regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr-490 of TrkA and was inactivated by 6-HITC in a redox-dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates.

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P(2) receptor, a carboxyl-terminal region of Galpha12 or Galpha13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P(2) receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P(2) receptors/G(12/13)-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P(2) receptor-mediated action in glioma cells.

GPR4 was initially identified as a receptor for sphingosylphosphorylcholine and lysophosphatidylcholine; however, lipid actions have not always been confirmed. Instead, ligand-independent actions have sometimes been observed in GPR4- and other OGR1 family receptor-expressing cells. Here, we examined the possible involvement of extracellular protons, which have recently been proposed as another ligand for GPR4. At pH 7.4, the epidermal growth factor-induced extracellular signal-regulated kinase activity was lower in GPR4-transfected RH7777 cells, in association with increased cAMP accumulation, than in vector-transfected cells. The serum response element (SRE)-driven transcriptional activity was also clearly higher in GPR4-expressing HEK293 cells than in vector-transfected cells at pH 7.4. These apparent ligand-independent actions were very small at alkalinic 7.8. The SRE activity was further increased by extracellular acidification in a manner dependent on the G13 protein/Rho signaling pathway in HEK293 cells expressing GPR4 or other OGR1 receptor family members. GPR4-expressing cells also showed a calcineurin-dependent nuclear factor of activated T cell (NFAT) promoter activation at pH 7.4, and this activity was further increased by pH below 7.2 in association with inositol phosphate production. In contrast to the cAMP and SRE responses, however, alkalinization to pH 7.8 hardly affected the high basal activity. Finally, the expression of GPR4 hardly modulated the sphingosylphosphorylcholine- or lysophosphatidylcholine-induced action. These results suggest that an extracellular proton play a role as a ligand in some of previously postulated ligand-independent actions through GPR4 receptors. Moreover, GPR4 may be a multi-functional receptor coupling to Gs, G13, and Gq/11 proteins in response to extracellular acidification.

We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. Beta1 Integrin were highly expressed on osteoblasts. Engagement of beta1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on osteoblasts. Rho was activated by beta1 stimulation in osteoblasts. Beta1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of beta1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in beta1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.

Sphingosine 1-phosphate (S1P) stimulates expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells. S1P-induced actions were associated with nuclear factor kappa-B activation and inhibited by pertussis toxin as well as by antisense oligonucleotides specific to S1P receptors, especially, S1P(3). S1P also stimulated endothelial nitric oxide synthase (eNOS) and its activation was markedly inhibited by the antisense oligonucleotide for the S1P(1) receptor rather than that for the S1P(3) receptor. The dose-response curve of S1P to stimulate adhesion molecule expression was shifted to the left in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin and the NOS inhibitor Nomega-nitro-l-arginine methyl ester. NO donor S-nitroso-N-acetylpenicillamine inhibited S1P-induced adhesion molecule expression. Moreover, tumor necrosis factor-alpha-induced adhesion molecule expression was markedly inhibited by S1P in a manner sensitive to inhibitors for PI3-K and NOS. These results suggest that S1P receptors are coupled to both stimulatory and inhibitory pathways for adhesion molecule expression. The stimulatory pathway involves nuclear factor kappa-B and inhibitory one does phosphatidylinositol 3-kinase and NOS.

Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Galpha13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Galpha13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Galpha13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells.

It is generally thought that Galpha(12) and Galpha(13)-induced responses are exclusively mediated by small G protein Rho. However, Galpha(12) and Galpha(13) elicit divergent cellular responses: phospholipase C-epsilon activation, phospholipase D activation, cytoskeletal change, oncogenic response, apoptosis, MAP kinase activation and Na/H-exchange activation. In addition to Rho activation through RhoGEF, it has been recently demonstrated that Galpha(12) and Galpha(13) interact with several proteins and regulate their activities. However, physiological importance of the interaction of Galpha(12) and Galpha(13) with these proteins has not fully established. I summarize the recent progress of Galpha(12) and Galpha(13)-mediated signaling cascade.

At the beginning, beta-arrestin is believed to regulate desensitization of G protein-coupled receptors. However, it becomes apparent that beta-arrestin is involved in not only regulation but also signal transduction by identification of many proteins interacting with beta-arrestin. So far, the model of regulation and signaling of G protein-coupled receptor is built on beta 2-adrenergic receptor. However, this model does not apply to all G protein-coupled receptors. The differential regulation and signaling between beta 1- and beta 2-adrenergic receptors is presented.

In rat neonatal myocytes, a constitutively active G alpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the G beta gamma-sequestering peptide beta ARK-ct increases the phenylephrine-induced cardiac injury, indicating that G beta gamma released from G(q) counteracts the G alpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by beta ARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that G beta gamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the G alpha(q)-induced cellular injury and apoptosis.

Extracellular information is translated into the cellular responses by various signal transduction systems such as G protein-coupled receptor-linked and protein tyrosine kinase receptor-mediated pathways. The changes or modifications of theses signaling proteins cause uncontrolled transformation or cell death. One of the features of the signaling molecules is that these proteins contain the domains or motifs for protein-protein or protein-lipid interactions governed by several amino acids. Therefore, these amino acids may become promising targets for the drug design. In contrast with intracellular molecules, the receptors located on cellular surface are still the best candidate for the drugs. The future analysis of cellular signal transduction system allows us to develop new drugs that cannot cause serious side effects in future.

Beta-arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. β-arrestin1 and β-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of β-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that β-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. β-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, β-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in β-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of β-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of β-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of β-arrestin2 KO mice is enhanced by MI, which is similar to that in β-arrestin2 KO mice. In contrast, the inflammation after MI in β-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that β-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the β-arrestin2 gene is knocked out. β-arrestin2 plays a protective role in MI-induced inflammation. [ABSTRACT FROM AUTHOR]