scholarly journals C5a-Stimulated Recruitment of β-Arrestin2 to the Nonsignaling 7-Transmembrane Decoy Receptor C5L2

2009 ◽  
Vol 14 (9) ◽  
pp. 1067-1075 ◽  
Author(s):  
Lambertus H.C. Van Lith ◽  
Julia Oosterom ◽  
Andrea Van Elsas ◽  
Guido J.R. Zaman
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Ligand Interaction ◽  
C5a Receptor ◽  
Decoy Receptor ◽  
Complementation Assay ◽  
Fragment Complementation

C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein—labeled β-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the β-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with β-arrestin was confirmed using a novel β-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of β-galactosidase, whereas β-arrestin2 was labeled with a corresponding deletion mutant of the enzyme. Stable transfection of the modified C5L2 and subsequent stimulation with C5a or C5a-desArg restored β-galactosidase activity in a dose-dependent manner. The subnanomolar potency of β-arrestin coupling in the β-galactosidase fragment complementation assay is in agreement with the affinity of the receptor-ligand interaction. C5L2 is the first example of a 7-transmembrane decoy receptor that couples to β-arrestin in a ligand-dependent manner. This observation supports the notion that G-protein-signaling and β-arrestin coupling can be 2 separate activities of 7-transmembrane receptors. Furthermore, the β-arrestin assays described in this article provide methods of screening for selective C5L2 modulators. ( Journal of Biomolecular Screening 2009:1067-1075)

181 THE POTENTIAL ROLE OF NON-GENOMIC PATHWAYS AND THEIR EFFECTS ON THE REGULATION OF CALBINDIN-D9k IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 189
Author(s):  
V. H. Dang ◽  
E.-B. Jeung
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Gh3 Cells ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Calbindin D9k ◽  
Protein Expressions ◽  
Genomic Effects

Calbindin-D9k (CaBP-9k), a cytosolic protein, is one of the members of the family of vitamin D-dependent calcium-binding proteins with high affinity for calcium. The previous in vitro studies indicated that this gene is controlled by 17β-estradiol (E2), a physiological estrogen, via both genomic (through its classical nuclear receptors) and non-genomic (through different cypoplasmic signals) mechanisms. In order to provide a better understanding in molecular events by which E2 exerts its actions in the regulation of CaBP-9k, we employed GH3 cells as an in vitro model to examine the possible non-genomic effects of E2 on the induction of CaBP-9k. GH3 cells were treated dose-dependently (10–5, 10–6, 10–7, 10–8, and 10–9 m) with E2-BSA, a membrane-impermeable E2 conjugated with BSA, for 24 h. To examine the time dependency, the cells were also exposed to a high concentration (10–6 m) of E2-BSA and harvested at various time points (5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h). Furthermore, in order to determine the potential involvement of non-genomic signaling pathways in E2-BSA-induced expression of CaBP-9k, several inhibitors also were employed, including ICI 182 780 for membrane estrogen receptor (ER) pathway, pertussis toxin (PTX) for G protein signaling, U0126 for ERK pathway, and wortmannin for Akt pathway. The non-genomic effects of E2-BSA on the induction of CaBP-9k mRNA and protein were determined by semi-quantitative RT-PCR and Western blotting, respectively. In a dose-dependent manner, administration with E2-BSA (10–6 m) induced the highest response of CaBP-9k at transcriptional (mRNA) level, whereas protein level of CaBP-9k peaked at E2-BSA concentration (10–7 m) at 24 h. In a time course, E2-BSA (10–6 m) exposure caused a significant increase in both CaBP-9k mRNA and protein expressions as early as 15 min and peaked at 24 h. Co-treatment with ICI 182 780 and PTX completely inhibited E2-BSA-induced CaBP-9k mRNA and protein expressions. Interestingly, although co-treatments with U0126 and/or wortmannin alone failed to attenuate the effects of E2-BSA, a combination of 2 inhibitors completely reversed E2-BSA-induced CaBP-9k expressions at both transcriptional (mRNA) and translational (protein) levels, suggesting their involvement in the regulation of CaBP-9k in GH3 cells. Taken together, these results demonstrate that various signaling pathways may be involved in E2-induced regulation of CaBP-9k in which membrane ER and G protein signaling pathways play a central role in non-genomic responses. Further in vitro experiments are required to elucidate additional details of the interaction of ERK and Akt pathways in the regulation of CaBP-9k in these cells, offering a new insight into the mode of E2 action in the pituitary gland of human and wildlife.

Mechanical and chemical activation of GPR68 (OGR1) probed with a genetically-encoded fluorescent reporter

2021 ◽  
Author(s):  
Alper D. Ozkan ◽  
Tina Gettas ◽  
Audrey Sogata ◽  
Wynn Phaychanpheng ◽  
Miou Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
Cancer Progression ◽  
Fluid Shear Stress ◽  
Fluorescent Protein ◽  
Chemical Activation ◽  
G Protein Signaling ◽  
Mechanical Stimuli ◽  
Protein Signaling ◽  
Fluorescent Reporter ◽  
Membrane Stretch

G-protein coupled receptor (GPCR) 68 (GPR68, or OGR1) couples extracellular acidifications and mechanical stimuli to G-protein signaling and plays important roles in vascular physiology, neuroplasticity, and cancer progression. Inspired by previous GPCR-based reporters, here, we inserted a cyclic permuted fluorescent protein into the third intracellular loop of GPR68 to create a genetically-encoded fluorescent reporter of GPR68 activation we call "iGlow". iGlow responds to known physiological GPR68 activators such as fluid shear stress and extracellular acidifications. In addition, iGlow responds to Ogerin, a synthetic GPR68-selective agonist, but not to a non-active Ogerin analog, showing the specificity of iGlow-mediated fluorescence signals. Flow-induced iGlow activation is not eliminated by pharmacological modulation of downstream G-protein signaling, disruption of actin filaments, or application of GsMTx4, an inhibitor of certain mechanosensitive ion channels activated by membrane stretch. Deletion of the conserved Helix 8, proposed to mediate mechanosensitivity in certain GPCRs, does not eliminate flow-induced iGlow activation. iGlow could be useful to investigate the contribution of GPR68-dependent signaling in health and disease.

Acyl-Protein Thioesterase 1 Functions in Palmitoylation/Depalmitoylation Cycles of G Proteins and Regulates Platelet Activation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1062-1062
Author(s):  
Louisa M. Dowal ◽  
James R. Dilks ◽  
Nathalie A. Fadel ◽  
Omozuanvbo R. Aisiku ◽  
Glenn Merrill-Skoloff ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Signaling Pathways ◽  
G Protein ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Granule Secretion ◽  
Proximal Signaling

Abstract Abstract 1062 Protein palmitoylation is a reversible post-translational modification that regulates both lipid-protein and protein-protein interactions. During the palmitoylation cycle, palmitoylation occurs through a thioester linkage to a cysteine residue. Depalmitoylation occurs primarily through cleavage of this bond by acyl-protein thioesterase 1 (APT1). We have previously demonstrated the presence of APT1 in platelets and showed that APT1 translocates to membranes in an activation-dependent manner. However, the function of APT1 in platelet activation is not known. To determine whether APT1 functions in platelet signal transduction we evaluated the effect of palmostatins, novel small molecule inhibitors of APT1, on platelet function. Palmostatins B and M both inhibited platelet aggregation and α -granule secretion induced through protease-activated receptor (PAR) 1 with an IC50 of 15 μM. To assess which signaling pathways were affected by APT1 inhibition, we screened palmostatins for their ability to inhibit activation induced by several agonists. Palmostatins blocked platelet aggregation induced by a PAR1 agonist, a PAR4 agonist, TxA2, or epinephrine. In contrast, palmostatins failed to inhibit aggregation induced by collagen, PMA, or ionophore. Palmostatins also inhibited α -granule exocytosis induced by a PAR1 agonist or TxA2, but not exocytosis induced by PMA or ionophore. These results suggested that palmostatins blocked proximal signaling events mediated through G protein coupled receptors (GPCRs). To evaluate this supposition, we tested the effect of palmostatin B on PAR1-mediated [Ca2+]i flux. Palmostatin B inhibited PAR1-induced Ca2+ signaling with and IC50 of 15 μM, the same concentration required for inhibition of platelet aggregation and α -granule secretion. We have recently described the platelet palmitoylome (Dowal et al., Blood, 118:e62-73) and found several components of the proximal G protein signaling pathway that are palmitoylated, including many Gα subunits. To directly assess the effect of APT1 inhibition on palmitoylation/depalmitoylation cycles on a target Gα subunit, we evaluated Gα q palmitoylation using acyl biotin exchange chemistry. Total Gα q palmitoylation decreased substantially with activation of platelets through PAR1. In the presence of palmostatin B, however, Gα q palmitoylation increased following PAR1 activation. These results demonstrate that Gα q is a substrate for APT1. Our studies demonstrate a role for palmitoylation/depalmitoylation cycles in proximal signaling pathways downstream of GPCRs and implicate APT1 as an essential regulator of G protein signaling in platelets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Correlation of Autophagosome Formation with Degradation and Endocytosis Arabidopsis Regulator of G-Protein Signaling (RGS1) through ATG8a

2019 ◽  
Vol 20 (17) ◽  
pp. 4190 ◽  
Author(s):  
Yue Jiao ◽  
Miroslav Srba ◽  
Jingchun Wang ◽  
Wenli Chen
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Autophagic Flux ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
In Planta ◽  
Autophagosome Formation ◽  
Root Cells ◽  
Ubiquitin Proteasome

Damaged or unwanted cellular proteins are degraded by either autophagy or the ubiquitin/proteasome pathway. In Arabidopsis thaliana, sensing of D-glucose is achieved by the heterotrimeric G protein complex and regulator of G-protein signaling 1 (AtRGS1). Here, we showed that starvation increases proteasome-independent AtRGS1 degradation, and it is correlated with increased autophagic flux. RGS1 promoted the production of autophagosomes and autophagic flux; RGS1-yellow fluorescent protein (YFP) was surrounded by vacuolar dye FM4-64 (red fluorescence). RGS1 and autophagosomes co-localized in the root cells of Arabidopsis and BY-2 cells. We demonstrated that the autophagosome marker ATG8a interacts with AtRGS1 and its shorter form with truncation of the seven transmembrane and RGS1 domains in planta. Altogether, our data indicated the correlation of autophagosome formation with degradation and endocytosis of AtRGS1 through ATG8a.

The Ca2+-sensing receptor couples to Gα12/13 to activate phospholipase D in Madin-Darby canine kidney cells

2004 ◽  
Vol 286 (1) ◽  
pp. C22-C30 ◽  
Author(s):  
Chunfa Huang ◽  
Kristine M. Hujer ◽  
Zhenzhen Wu ◽  
R. Tyler Miller
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Phospholipase D ◽  
Pertussis Toxin ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: Gαi to inhibit the activity of adenylyl cyclase and activate ERK, Gαq to stimulate phospholipase C and phospholipase A2, and Gβγ to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to Gα12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known Gα12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaRWT) or the nonfunctional mutant CaRR796W as a negative control, prelabeled these cells with [3H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [3H]phosphatidylethanol (PEt). The formation of [3H]PEt increased in a time-dependent manner in the cells that overexpress the CaRWT but not the CaRR796W. Treatment of the cells with C3 exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of Gα12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate Gαi or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for Gαi and Gαq, and a p115RhoGEF construct containing the RGS domain for Gα12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWT inhibited extracellular Ca2+-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to Gα12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of Gαi and Gαq. This suggests that the CaR may regulate cytoskeleton via Gα12/13, Rho, and PLD.

scholarly journals Interactome Analysis Reveals Regulator of G Protein Signaling 14 (RGS14) is a Novel Calmodulin (CaM) Effector in Mouse Brain

2018 ◽  
Author(s):  
Paul R. Evans ◽  
Kyle J. Gerber ◽  
Eric B. Dammer ◽  
Duc M. Duong ◽  
Devrishi Goswami ◽  
...  
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Mouse Brain ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling

AbstractRegulator of G Protein Signaling 14 (RGS14) is a complex scaffolding protein with an unusual domain structure that allows it to integrate G protein and mitogen-activated protein kinase (MAPK) signaling pathways. RGS14 mRNA and protein are enriched in brain tissue of rodents and primates. In the adult mouse brain, RGS14 is predominantly expressed in postsynaptic dendrites and spines of hippocampal CA2 pyramidal neurons where it naturally inhibits synaptic plasticity and hippocampus-dependent learning and memory. However, the signaling proteins that RGS14 natively interacts with in neurons to regulate plasticity are unknown. Here, we show that RGS14 exists as a component of a high molecular weight protein complex in brain. To identify RGS14 neuronal interacting partners, endogenous RGS14 immunoprecipitated from mouse brain was subjected to mass spectrometry and proteomic analysis. We find that RGS14 interacts with key postsynaptic proteins that regulate neuronal plasticity. Gene ontology analysis reveals that the most enriched RGS14 interacting proteins have functional roles in actin-binding, calmodulin(CaM)-binding, and CaM-dependent protein kinase (CaMK) activity. We validate these proteomics findings using biochemical assays that identify interactions between RGS14 and two previously unknown binding partners: CaM and CaMKII. We report that RGS14 directly interacts with CaM in a calcium-dependent manner and is phosphorylated by CaMKII in vitro. Lastly, we detect that RGS14 associates with CaMKII and with CaM in hippocampal CA2 neurons by proximity ligation assays in mouse brain sections. Taken together, these findings demonstrate that RGS14 is a novel CaM effector and CaMKII phosphorylation substrate thereby providing new insight into cellular mechanisms by which RGS14 controls plasticity in CA2 neurons.

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