Agonists of the MAS-related gene (Mrgs) orphan receptors as novel mediators of mast cell-sensory nerve interactions

IgE-dependent activation of mast cell activation is often associated with symptoms attributed to activation of sensory nerves. Depending on the tissues involved such symptoms include itching, sneezing, irritation, vasodilation, and reflex secretions. In the present study, we hypothesize that sensory neuroactive mediators released from mast cells may include agonists of recently discovered orphan receptors referred to as sensory nerve specific receptors or products of mas related genes. HEK-293 cells expressing MrgC11 receptors and wild-type HEK-293 cells were loaded with the calcium indicator Fura-2. A known stimulant of MrgC11 receptors the RF-amide, neuropeptide FF, evoked a rapid increase in cytosolic calcium in the MrgC11 expressing cells but not in the wild-type HEK-293 cells. IgE-dependent stimulation of either rat basophilic leukemia-2H3 cells (RBL-2H3 cells) or mouse bone marrow-derived mast cells, released a substance(s) that stimulated increases in cytosolic calcium in the MrgC11 expressing cells that far exceeded that seen in control cells. RT-PCR revealed that both mouse mast cells and RBL-2H3 cells express the RF-amide precursor gene proneuropeptide FF (A). Immunohistochemical analysis demonstrated RF-amide immunoreactivity in mouse skin mast cells in situ and in mast cells isolated from mouse skin. These data support the hypothesis that agonists of certain sensory nerve specific receptors or mas related genes may participate in mast cell sensory nerve interactions.     

Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis

A class of small molecules displaying comparable activities with peptide ligands BAM22 and corticostatin-14 at both the human and rhesus monkey MrgX1 and MrgX2 receptors, respectively, was discovered. A comparative study to compare solid-phase and solution-phase chemistries for the efficient synthesis of the active class, tetracyclic benzimidazoles, was undertaken. The solid-phase chemistry was found to be superior both for the synthesis of analogs and for the synthesis of gram quantities.     

Sequence and Functional Analysis of Cloned Guinea Pig and Rat Serotonin 5-HT1D Receptors: Common Pharmacological Features Within the 5-HT1D Receptor Subfamily

Abstract: This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)_1D receptor sites. Guinea pig, rat, and mouse 5-HT_1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT_1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13–27%) in the N-terminal extracellular region of these 5-HT_1D receptors. Guinea pig and rat 5-HT_1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT_1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT_1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT_1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT_1D receptor-mediated antagonist potency for methiothepin and the 5-HT₂ receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT_1D receptor subtype seems, unlike the 5-HT_1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

Mas-related gene X2 (MrgX2) is a novel G protein-coupled receptor for the antimicrobial peptide LL-37 in human mast cells: resistance to receptor phosphorylation, desensitization, and internalization

Human LL-37 is a multifunctional antimicrobial peptide that promotes inflammation, angiogenesis, wound healing, and tumor metastasis. Most effects of LL-37 are mediated via the activation of the cell surface G protein-coupled receptor FPR2 on leukocytes and endothelial cells. Although LL-37 induces chemotaxis, degranulation, and chemokine production in mast cells, the receptor involved and the mechanism of its regulation remain unknown. MrgX2 is a member of Mas-related genes that is primarily expressed in human dorsal root ganglia and mast cells. We found that a human mast cell line LAD2 and CD34(+) cell-derived primary mast cells, which natively express MrgX2, responded to LL-37 for sustained Ca(2+) mobilization and substantial degranulation. However, an immature human mast cell line, HMC-1, that lacks functional MrgX2 did not respond to LL-37. shRNA-mediated knockdown of MrgX2 in LAD2 mast cell line and primary CD34(+) cell-derived mast cells caused a substantial reduction in LL-37-induced degranulation. Furthermore, mast cell lines stably expressing MrgX2 responded to LL-37 for chemotaxis, degranulation, and CCL4 production. Surprisingly, MrgX2 was resistant to LL-37-induced phosphorylation, desensitization, and internalization. In addition, shRNA-mediated knockdown of the G protein-coupled receptor kinases (GRK2 and GRK3) had no effect on LL-37-induced mast cell degranulation. This study identified MrgX2 as a novel G protein-coupled receptor for the antibacterial peptide LL-37 and demonstrated that unlike most G protein-coupled receptors it is resistant to agonist-induced receptor phosphorylation, desensitization, and internalization.     

Characterisation of AmphiAmR11, an Amphioxus (Branchiostoma floridae) D2-Dopamine-Like G Protein-Coupled Receptor

The evolution of the biogenic amine signalling system in vertebrates is unclear. However, insights can be obtained from studying the structures and signalling properties of biogenic amine receptors from the protochordate, amphioxus, which is an invertebrate species that exists at the base of the chordate lineage. Here we describe the signalling properties of AmphiAmR11, an amphioxus (Branchiostoma floridae) G protein-coupled receptor which has structural similarities to vertebrate α₂-adrenergic receptors but which functionally acts as a D₂ dopamine-like receptor when expressed in Chinese hamster ovary -K1 cells. AmphiAmR11 inhibits forskolin-stimulated cyclic AMP levels with tyramine, phenylethylamine and dopamine being the most potent agonists. AmphiAmR11 also increases mitogen-activated protein kinase activity and calcium mobilisation, and in both pathways, dopamine was found to be more potent than tyramine. Thus, differences in the relative effectiveness of various agonists in the different second messenger assay systems suggest that the receptor displays agonist-specific coupling (biased agonism) whereby different agonists stabilize different conformations of the receptor which lead to the enhancement of one signalling pathway over another. The present study provides insights into the evolution of α₂-adrenergic receptor signalling and support the hypothesis that α₂-adrenergic receptors evolved from D₂-dopamine receptors. The AmphiAmR11 receptor may represent a transition state between D₂-dopamine receptors and α₂-adrenergic receptors.     

The prostaglandin EP4 receptor is a master regulator of the expression of PGE2 receptors following inflammatory activation in human monocytic cells

Prostaglandin E₂ (PGE₂) is responsible for inflammatory symptoms. However, PGE₂ also suppresses pro-inflammatory cytokine production. There are at least 4 subtypes of PGE₂ receptors, EP1–EP4, but it is unclear which of these specifically control cytokine production. The aim of this study was to determine which of the different receptors, EP1R–EP4R modulate production of tumor necrosis factor-α (TNF-α) in human monocytic cells.Human blood, or the human monocytic cell line THP-1 were stimulated with LPS. The actions of PGE₂, alongside selective agonists of EP1–EP4 receptors, were assessed on LPS-induced TNF-α, IL-1β and IL-10 release. The expression profiles of EP2R and EP4R in monocytes and THP-1 cells were characterised by RT-qPCR. In addition, the production of cytokines was evaluated following knockdown of the receptors using siRNA and over-expression of the receptors by transfection with constructs.PGE₂ and also EP2 and EP4 agonists (but not EP1 or EP3 agonists) suppressed TNF-α production in blood and THP-1 cells. LPS also up regulated expression of EP2R and EP4R but not EP1 or EP3. siRNA for either EP2R or EP4R reversed the suppressive actions of PGE₂ on cytokine production and overexpression of EP2R and EP4R enhanced the suppressive actions of PGE₂.This indicates that PGE₂ suppression of TNF-α by human monocytic cells occurs via EP2R and EP4R expression. However EP4Rs also control their own expression and that of EP2 whereas the EP2R does not affect EP4R expression. This implies that EP4 receptors have an important master role in controlling inflammatory responses.     

MrgX2 is a high potency cortistatin receptor expressed in dorsal root ganglion

MrgX2 is a recently identified orphan G-protein-coupled receptor whose ligand and physiological function were unknown. Here we describe cortistatin, a neuropeptide for which no specific receptor has been identified previously, as a high potency ligand at MrgX2. Cortistatin has several biological functions including roles in sleep regulation, locomotor activity, and cortical function. Using a "reverse pharmacology" approach, we have identified a number of additional cyclic peptide agonists for MrgX2, determined their rank order of potency, and demonstrated that this receptor has a pharmacological profile distinct from the other characterized members of the Mrg (Mas-related genes) family. In MrgX2-expressing cells, cortistatin-stimulated increases in intracellular Ca2+ but had no effect on basal or forskolin-stimulated cAMP levels, suggesting that this receptor is Gq-coupled. Immunohistochemical and quantitative PCR studies show MrgX2 to have a limited expression profile, both peripheral and within the central nervous system, with highest levels in dorsal root ganglion.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

Characterizing Ligand-Gated Ion Channel Receptors with Genetically Encoded Ca++ Sensors

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca²⁺ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT_3A serotonin receptors and a chimera of human α7/mouse 5-HT_3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.     

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M₁-, M₃-, and M₅-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70 ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M₁- and M₅-acetylcholine receptors and the amplitude of these signals was larger at the M₁-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M₁-, M₃- and M₅-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation.     

Design,synthesis and pharmacological evaluation of novel naphthalenic derivatives as selective MT1 melatoninergic ligands

Novel heterodimer analogues of melatonin were synthesized, when agomelatine (1) and various aryl units are linked via a linear alkyl chain through the methoxy group. The compounds were tested for their actions at melatonin receptors. Several of these ligands are MT₁-selective with nanomolar or subnanomolar affinity. In addition, while most of the derivatives behave as partial agonists on one or both receptor subtypes, N-[2-(7-{4-[6-(1-methoxycarbonylethyl)naphthalen-2-yloxy]butoxy}naphthalen-1-yl)ethyl]acetamide (36), a subnanomolar MT₁ ligand with an 11-fold preference over MT₂ receptors, is a full antagonist on both receptors. Our results also confirm that the selectivity seen for the MT₁ receptor arises predominantly from steric factors and is not a consequence of the bridging of melatonin receptor dimers.     

Pharmacological Characterisation of [35S]-GTPγS Binding to Chinese Hamster Ovary Cell Membranes Stably Expressing Cloned Human 5-HT1D Receptor Subtypes

Abstract

[³⁵S]-GTPγS binding has been used to study the function of cloned human 5-HT_1D receptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [³⁵S]-GTPγS binding to membranes from cells expressing 5-HT_1Dα or 5-HT_1Dβ receptors. In membranes containing 5-HT_1Dβ receptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT > 5-HT > metergoline > sumatriptan > yohimbine > 8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT_1Dβ receptors was potently antagonised by methiothepin (pA₂ 8.9 ± 0.1). The overall pharmacological profile for the human 5-HT_1Dβ receptor, defined using [³⁵S]-GTPγS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [³⁵S]-GTPγS binding to membranes containing 5-HT_1Dα or 5-HT_1Dβ receptors, suggesting that these compounds show negative efficacy at 5-HT_1D receptor subtypes. The data show that [³⁵S]-GTPγS binding is a suitable method for studying the interaction between cloned human 5-HT_1D receptors and G-proteins.     

Receptor-Galpha fusion proteins as a tool for ligand screening

We have prepared fusion proteins of muscarinic M1-M5 receptors with alpha subunits of G proteins Gi1, Gi2, Gs, G11, G16 and chimera of G protein alpha subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M2-Gi1alpha and M4-Gi1alpha, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [35S]GTPgammaS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of Gq/G11-coupled receptors such as M1-G11alpha, M3-G11alpha, and M5-G11alpha. The effect of agonists could be observed for fusion proteins with G16alpha of muscarinic M1, M2 and adrenergic beta2 receptors, but the extent of the effect was much less than that for fusion proteins with Gi1alpha of Gi/Go-coupled receptors. Fusion proteins of M1 receptors with Gi1alpha or chimera of G16alpha and Gi2alpha were also not effective in detecting the action of agonists.     

Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

The human MrgX3 gene, belonging to the mrgs/SNSRs (mas related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.     

Novel GPCR Screening Approach: Indirect Identification of S1P Receptor Agonists in Antagonist Screening Using a Calcium Assay

To elucidate the physiological function of sphingosine 1-phosphate receptors 1–3 (S1P_1?3) we aimed to identify selective ligands for these GPCRs. S1P₂ and S1P₃ are coupled to G_q, and are, therefore, linked to the phospholipase C/IP₃/calcium pathway. S1P₁ is solely coupled to G_i and was artificially linked to calcium signaling by coexpression of Gα 16. The three receptors desensitized on challenge of cells with an agonist (i.e., agonists appeared as antagonists in a second calcium measurement). We screened a compound library for inhibitors of S1P-stimulated calcium signals, and we could identify agonists and antagonists with a single measurement. Agonism and antagonism were confirmed by recording compound-and S1P-induced calcium signals from the same assay well. For the three receptors, we found a reciprocal correlation of agonism and “apparent” antagonism of agonists. In addition, agonists indirectly discovered by this approach do not promote calcium mobilization through endogenous GPCRs.     

Synthesis and binding study of certain 6-arylalkanamides as molecular probes for cannabinoid receptor subtypes

Tetrahydrocannabinol and other mixed cannabinoid (CB) receptors CB₁/CB₂ receptor agonists are well established to elicit antinociceptive effects and psychomimetic actions, however, their potential for abuse have dampened enthusiasm for their therapeutic development. In an effort to refine a semi-rigid structural framework for CB₂ receptors binding, we designed novel compounds based on aromatic moiety and flexible linker with various amides mimicking the outlook of the endogenous anandamide which could provide as CB₂ receptor ligand. In this direction, we developed and synthesized new aryl or arylidene hexanoic acid amides and aryl alkanoic acid diamide carrying different head groups. These new compounds were tested for their affinities for human recombinant CB receptors CB₁ and CB₂ and fatty acid amide hydrolase. Although, the preliminary screening of these compounds demonstrated weak binding activity towards CB receptor subtypes at 10 µmole, yet this template still could serve up as probes for further optimization and development of affinity ligand for CB receptors.     

Molecular and pharmacological characterization of two D(1)-like dopamine receptors in the Lyme disease vector, Ixodes scapularis

Advancements in tick neurobiology may impact the development of acaricides to control those species that transmit human and animal diseases. Here, we report the first cloning and pharmacological characterization of two neurotransmitter binding G protein-coupled receptors in the Lyme disease (blacklegged) tick, Ixodes scapularis. The genes IscaGPRdop1 and IscaGPRdop2 were identified in the I. scapularis genome assembly and predicted as orthologs of previously characterized D₁-like dopamine receptors in the fruit fly Drosophila melanogaster and honeybee Apis mellifera. Heterologous expression in HEK 293 cells demonstrated that each receptor functioned as a D₁-like dopamine receptor because significant increases in levels of intracellular cyclic adenosine monophosphate (cAMP) were detected following dopamine treatment. Importantly, the receptors were distinct in their pharmacological properties regarding concentration-dependent response to dopamine, constitutive activity, and response to other biogenic amines. Exposure to a variety of dopamine receptor agonists and antagonists further demonstrated a D₁-like pharmacology of these dopamine receptors and highlighted their differential activities in vitro.     

Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P₂ and S1P₃ receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P₃ receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P₃R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P₂R and S1P₃R stimulation using Smad-independent pathways.     

Prostanoid Receptors Involved in Regulation of the Beating Rate of Neonatal Rat Cardiomyocytes

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F_2α and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD₂ and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP₁ and EP₃ receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP₁ antagonist). Butaprost (a selective prostanoid EP₂ receptor agonist), misoprostol (a prostanoid EP₂ and EP₃ receptor agonist), 11-deoxy-PGE₁ (a prostanoid EP₂, EP₃ and EP₄ receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP₁ receptors are involved in positive regulation of the beating rate. Prostanoid EP₁ receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP₁ and EP₁ receptors (which positively regulate the spontaneous beating rate).