cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Receptor Activation

AbstractThe kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to β-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor–biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.

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Role of Ca2+ in Mediating Plant Responses to Extracellular ATP and ADP

International Journal of Molecular Sciences ◽

10.3390/ijms19113590 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3590 ◽

Cited By ~ 8

Author(s):

Greg Clark ◽

Stanley Roux

Keyword(s):

Cytosolic Calcium ◽

Defense Responses ◽

Receptor Activation ◽

Extracellular Atp ◽

Extracellular Nucleotides ◽

Nucleoside Triphosphate ◽

Plant Responses ◽

Downstream Signaling ◽

Nucleoside Triphosphate Diphosphohydrolase

Among the most recently discovered chemical regulators of plant growth and development are extracellular nucleotides, especially extracellular ATP (eATP) and extracellular ADP (eADP). Plant cells release ATP into their extracellular matrix under a variety of different circumstances, and this eATP can then function as an agonist that binds to a specific receptor and induces signaling changes, the earliest of which is an increase in the concentration of cytosolic calcium ([Ca2+]cyt). This initial change is then amplified into downstream-signaling changes that include increased levels of reactive oxygen species and nitric oxide, which ultimately lead to major changes in the growth rate, defense responses, and leaf stomatal apertures of plants. This review presents and discusses the evidence that links receptor activation to increased [Ca2+]cyt and, ultimately, to growth and diverse adaptive changes in plant development. It also discusses the evidence that increased [Ca2+]cyt also enhances the activity of apyrase (nucleoside triphosphate diphosphohydrolase) enzymes that function in multiple subcellular locales to hydrolyze ATP and ADP, and thus limit or terminate the effects of these potent regulators.

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Vav Family Proteins Couple to Diverse Cell Surface Receptors

Molecular and Cellular Biology ◽

10.1128/mcb.20.17.6364-6373.2000 ◽

2000 ◽

Vol 20 (17) ◽

pp. 6364-6373 ◽

Cited By ~ 106

Author(s):

Sheri L. Moores ◽

Laura M. Selfors ◽

Jessica Fredericks ◽

Timo Breit ◽

Keiko Fujikawa ◽

...

Keyword(s):

Growth Factor ◽

Cell Surface ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Receptor Activation ◽

Cell Surface Receptors ◽

Nucleotide Exchange ◽

Surface Receptors ◽

Downstream Signaling

ABSTRACT Vav proteins are guanine nucleotide exchange factors for Rho family GTPases which activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations. Vav proteins contain several protein binding domains which can link cell surface receptors to downstream signaling proteins. Vav1 is expressed exclusively in hematopoietic cells and tyrosine phosphorylated in response to activation of multiple cell surface receptors. However, it is not known whether the recently identified isoforms Vav2 and Vav3, which are broadly expressed, can couple with similar classes of receptors, nor is it known whether all Vav isoforms possess identical functional activities. We expressed Vav1, Vav2, and Vav3 at equivalent levels to directly compare the responses of the Vav proteins to receptor activation. Although each Vav isoform was tyrosine phosphorylated upon activation of representative receptor tyrosine kinases, integrin, and lymphocyte antigen receptors, we found unique aspects of Vav protein coupling in each receptor pathway. Each Vav protein coprecipitated with activated epidermal growth factor and platelet-derived growth factor (PDGF) receptors, and multiple phosphorylated tyrosine residues on the PDGF receptor were able to mediate Vav2 tyrosine phosphorylation. Integrin-induced tyrosine phosphorylation of Vav proteins was not detected in nonhematopoietic cells unless the protein tyrosine kinase Syk was also expressed, suggesting that integrin activation of Vav proteins may be restricted to cell types that express particular tyrosine kinases. In addition, we found that Vav1, but not Vav2 or Vav3, can efficiently cooperate with T-cell receptor signaling to enhance NFAT-dependent transcription, while Vav1 and Vav3, but not Vav2, can enhance NFκB-dependent transcription. Thus, although each Vav isoform can respond to similar cell surface receptors, there are isoform-specific differences in their activation of downstream signaling pathways.

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Effects of Prednisolone on Serum and Tissue Fluid IGF-I Receptor Activation and Post-Receptor Signaling in Humans

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2017-00696 ◽

2017 ◽

Vol 102 (11) ◽

pp. 4031-4040 ◽

Cited By ~ 9

Author(s):

Nilani Ramshanker ◽

Maiken Aagaard ◽

Rikke Hjortebjerg ◽

Thomas Schmidt Voss ◽

Niels Møller ◽

...

Keyword(s):

Messenger Rna ◽

Protein A ◽

Receptor Activation ◽

Tissue Fluid ◽

Igf Binding Proteins ◽

Downstream Signaling ◽

Specific Effects ◽

Igf I ◽

Underlying Mechanisms ◽

Double Blinded

Abstract Context Short-term glucocorticoid exposure increases serum insulinlike growth factor I (IGF-I) concentrations but antagonizes IGF-I tissue signaling. The underlying mechanisms remain unknown. Objective To identify at which levels glucocorticoid inhibits IGF-I signaling. Design and Methods Nineteen healthy males received prednisolone (37.5 mg/d) and placebo for 5 days in a randomized, double-blinded, placebo-controlled crossover study. Serum was collected on days 1, 3, and 5, and abdominal skin suction blister fluid (SBF; ~interstitial fluid) was taken on day 5 (n = 9) together with muscle biopsy specimens (n = 19). The ability of serum and SBF to activate the IGF-I receptor (IGF-IR) (bioactive IGF) and its downstream signaling proteins was assessed using IGF-IR–transfected cells. Results Prednisolone increased IGF-I concentrations and bioactive IGF in serum (P ≤ 0.001) but not in SBF, which, compared with serum, contained less bioactive IGF (~28%) after prednisolone (P < 0.05). This observation was unexplained by SBF concentrations of IGFs and IGF-binding proteins (IGFBPs) 1 to 4. However, following prednisolone treatment, SBF contained less IGFBP-4 fragments (P < 0.05) generated by pregnancy-associated plasma protein A (PAPP-A). Concomitantly, prednisolone increased SBF levels of stanniocalcin 2 (STC2) (P = 0.02) compared with serum. STC2 blocks PAPP-A from cleaving IGFBP-4. Finally, prednisolone suppressed post–IGF-IR signaling pathways at the level of insulin receptor substrate 1 (P < 0.05) but did not change skeletal muscle IGF-IR, IGF-I, or STC2 messenger RNA. Conclusion Prednisolone increased IGF-I concentrations and IGF bioactivity in serum but not in tissue fluid. The latter may relate to a STC2-mediated inhibition of PAPP-A in tissue fluids. Furthermore, prednisolone induced post–IGF-IR resistance. Thus, glucocorticoid may exert distinct, compartment-specific effects on IGF action.

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Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles

Scientific Reports ◽

10.1038/s41598-020-75108-3 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Blanca V. Rodriguez ◽

Meta J. Kuehn

Keyword(s):

Staphylococcus Aureus ◽

Nucleic Acids ◽

Immune Modulation ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Receptor Activation ◽

Host Cells ◽

Downstream Signaling ◽

Host Immune Responses ◽

Rna And Dna

Abstract Bacterial-derived RNA and DNA can function as ligands for intracellular receptor activation and induce downstream signaling to modulate the host response to bacterial infection. The mechanisms underlying the secretion of immunomodulatory RNA and DNA by pathogens such as Staphylococcus aureus and their delivery to intracellular host cell receptors are not well understood. Recently, extracellular membrane vesicle (MV) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. S. aureus produce membrane-bound, spherical, nano-sized, MVs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. Here we show that S. aureus secretes RNA and DNA molecules that are mostly protected from degradation by their association with MVs. Importantly, we demonstrate that MVs can be delivered into cultured macrophage cells and subsequently stimulate a potent IFN-β response in recipient cells via activation of endosomal Toll-like receptors. These findings advance our understanding of the mechanisms by which bacterial nucleic acids traffic extracellularly to trigger the modulation of host immune responses.

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Evidence for Activation of Toll-Like Receptor and Receptor for Advanced Glycation End Products in Preterm Birth

Mediators of Inflammation ◽

10.1155/2010/490406 ◽

2010 ◽

Vol 2010 ◽

pp. 1-10 ◽

Cited By ~ 23

Author(s):

Taketoshi Noguchi ◽

Toshiyuki Sado ◽

Katsuhiko Naruse ◽

Hiroshi Shigetomi ◽

Akira Onogi ◽

...

Keyword(s):

Preterm Birth ◽

Advanced Glycation End Products ◽

Expression Profiles ◽

Receptor Activation ◽

Advanced Glycation ◽

Specific Expression ◽

Downstream Signaling ◽

Glycation End Products ◽

End Products ◽

Rage Expression

Objective. Individuals with inflammation have a myriad of pregnancy aberrations including increasing their preterm birth risk. Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE) and their ligands were all found to play a key role in inflammation. In the present study, we reviewed TLR and RAGE expression, their ligands, and signaling in preterm birth.Research Design and Methods. A systematic search was performed in the electronic databases PubMed and ScienceDirect up to July 2010, combining the keywords “preterm birth,” “TLR”, “RAGE”, “danger signal”, “alarmin”, “genomewide,” “microarray,” and “proteomics” with specific expression profiles of genes and proteins.Results. This paper provides data on TLR and RAGE levels and critical downstream signaling events including NF-kappaB-dependent proinflammatory cytokine expression in preterm birth. About half of the genes and proteins specifically present in preterm birth have the properties of endogenous ligands “alarmin” for receptor activation. The interactions between the TLR-mediated acute inflammation and RAGE-mediated chronic inflammation have clear implications for preterm birth via the TLR and RAGE system, which may be acting collectively.Conclusions. TLR and RAGE expression and their ligands, signaling, and functional activation are increased in preterm birth and may contribute to the proinflammatory state.

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Diminution of feeding response in Helicoverpa armigera by inhibition or silencing of sugar gustatory receptor

10.1101/599324 ◽

2019 ◽

Author(s):

Aniruddha R. Agnihotri ◽

Sanyami S. Zunjarrao ◽

Mukta Nagare ◽

Rakesh S. Joshi

Keyword(s):

Helicoverpa Armigera ◽

Feeding Rate ◽

Receptor Activation ◽

Inhibitory Effect ◽

Gustatory Receptor ◽

Feeding Response ◽

Downstream Signaling ◽

Helicoverpa Armígera ◽

Novel Strategy

ABSTRACTGustatory receptor (GR) is one of the essential chemosensory molecules in Lepidopteran pests. GR is involved in sensing several canonical tastes which in turn regulate the diverse behavioral and physiological responses of these insects. In this article, we have evaluated the alteration in feeding response of Helicoverpa armigera by blocking and silencing of sugar-sensing gustatory receptor 9 (HarmGr9). Sf9 cells based assay showed that glucose analogue, Miglitol, can bind to the expressed HarmGr9. This binding might lead to an inhibition of receptor activation and downstream signaling, indicated by reduced intracellular Ca2+ fluorescence. Further, the in-vivo study illustrated the feeding rate reduced on a diet containing miglitol as compared to the larvae fed on the artificial diet. Reduction in feeding rate was prolonged in insects fed on the miglitol containing diet even after switching to the control glucose diet. Competitive cell-based assay and feeding assay, using equimolar glucose and miglitol, also showed an inhibitory effect on HarmGr-9 activation and insect feeding rate. We have observed similar feeding rate reduction in HarmGr9 knockdown in H. armigera larvae. We believe this unique approach of H. armigera feeding response inhibition by blocking the sugar receptor can be further used to develop a novel strategy for agricultural pest management.

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Adrenergic receptors in osteoarthritic and rheumatoid synovial fibroblasts: Identification of β3 as novel player to modulate IL-6 and p38 activation

10.21203/rs.3.rs-299542/v1 ◽

2021 ◽

Author(s):

Ding Liang ◽

Torsten Lowin ◽

Xinkun Cheng ◽

Tim Classen ◽

Georg Pongratz

Keyword(s):

Synovial Tissue ◽

Adrenergic Receptors ◽

Adrenergic Receptor ◽

Synovial Fibroblasts ◽

Receptor Activation ◽

Adrenergic System ◽

Downstream Signaling ◽

Therapeutic Opportunity ◽

Severity Of The Disease ◽

And Function

Abstract Background: Rheumatoid arthritis (RA) is influenced by the activity of the sympathetic nervous system (SNS). In animal models of RA, the SNS promotes severity of the disease and its manipulation modulates experimental arthritis depending on timing of the intervention. Synovial fibroblasts (SF) are major contributors to RA pathology but their modulation by the SNS has been rarely investigated. In this study we assessed the expression and function of adrenergic receptors in RA and osteoarthritis (OA) synovial fibroblasts and investigated their downstream signaling. Methods: We used western blot and quantitative PCR (qPCR) to determine protein and mRNA of adrenergic receptors in OASF/RASF. Furthermore we determined α1a and β2 protein in synovial tissue by immunofluorescence. ELISA was employed to determine IL-6 production. p38 kinase activation and translocation was analyzed by cell-based ELISA and immunofluorescence.Results: We detected α1a, α2b, β1, β2 and β3 protein in OASF/RASF and α1a and β2 protein in synovial tissue of OA and RA patients. The pro-inflammatory cytokines IFN-γ and TNF downregulated β3 adrenergic receptor. Activation of α1a, α2b, β2 and β3 increased production of TNF-induced IL-6 which was inhibited by specific antagonists. Furthermore, β3 agonism enhanced p38 phosphorylation and translocation to the nucleus.Conclusion: Among a comprehensive characterization of the adrenergic system of OASF/ RASF, we report for the first time β3 expression and demonstrated that this adrenergic receptor participates in the inflammatory response of synovial fibroblasts. Therefore, modulation of β3 might pose a new therapeutic opportunity to modulate synovial fibroblast function in patients with RA.

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Roles of sphingosine-1-phosphate signaling in cancer

Cancer Cell International ◽

10.1186/s12935-019-1014-8 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 13

Author(s):

Peng Wang ◽

Yonghui Yuan ◽

Wenda Lin ◽

Hongshan Zhong ◽

Ke Xu ◽

...

Keyword(s):

Cell Fate ◽

Cancer Progression ◽

Therapeutic Potential ◽

Receptor Activation ◽

Signaling Cascades ◽

Lipid Mediator ◽

Downstream Signaling ◽

Sphingosine 1 Phosphate ◽

Sphingosine Kinases ◽

And Migration

AbstractThe potent pleiotropic lipid mediator sphingosine-1-phosphate (S1P) participates in numerous cellular processes, including angiogenesis and cell survival, proliferation, and migration. It is formed by one of two sphingosine kinases (SphKs), SphK1 and SphK2. These enzymes largely exert their various biological and pathophysiological actions through one of five G protein-coupled receptors (S1PR1–5), with receptor activation setting in motion various signaling cascades. Considerable evidence has been accumulated on S1P signaling and its pathogenic roles in diseases, as well as on novel modulators of S1P signaling, such as SphK inhibitors and S1P agonists and antagonists. S1P and ceramide, composed of sphingosine and a fatty acid, are reciprocal cell fate regulators, and S1P signaling plays essential roles in several diseases, including inflammation, cancer, and autoimmune disorders. Thus, targeting of S1P signaling may be one way to block the pathogenesis and may be a therapeutic target in these conditions. Increasingly strong evidence indicates a role for the S1P signaling pathway in the progression of cancer and its effects. In the present review, we discuss recent progress in our understanding of S1P and its related proteins in cancer progression. Also described is the therapeutic potential of S1P receptors and their downstream signaling cascades as targets for cancer treatment.

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The emerging role of the fibroblast growth factor-23–klotho axis in renal regulation of phosphate homeostasis

Journal of Endocrinology ◽

10.1677/joe-07-0095 ◽

2007 ◽

Vol 194 (1) ◽

pp. 1-10 ◽

Cited By ~ 145

Author(s):

Mohammed S Razzaque ◽

Beate Lanske

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Fibroblast Growth Factor 23 ◽

Ion Homeostasis ◽

Experimental Studies ◽

Receptor Activation ◽

Fibroblast Growth ◽

Phosphate Homeostasis ◽

Downstream Signaling

Normal mineral ion homeostasis is tightly controlled by numerous endocrine factors that coordinately exert effects on intestine, kidney, and bone to maintain physiological balance. The importance of the fibroblast growth factor (FGF)-23–klotho axis in regulating mineral ion homeostasis has been proposed from recent research observations. Experimental studies suggest that 1) FGF23 is an important in vivo regulator of phosphate homeostasis, 2) FGF23 acts as a counter regulatory hormone to modulate the renal 1α-hydroxylase and sodium–phosphate cotransporter activities, 3) there is a trend of interrelationship between FGF23 and parathyroid hormone activities, 4) most of the FGF23 functions are conducted through the activation of FGF receptors, and 5) such receptor activation needs klotho, as a cofactor to generate downstream signaling events. These observations clearly suggest the emerging roles of the FGF23–klotho axis in maintaining mineral ion homeostasis. In this brief article, we will summarize how the FGF23–klotho axis might coordinately regulate normal mineral ion homeostasis, and how their abnormal regulation could severely disrupt such homeostasis to induce disease pathology.

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