scholarly journals Endogenous modification of the chemoattractant CXCL5 alters receptor usage and enhances its activity toward neutrophils and monocytes

2021 ◽  
Vol 14 (673) ◽  
pp. eaax3053
Author(s):  
Mieke Metzemaekers ◽  
Anneleen Mortier ◽  
Alessandro Vacchini ◽  
Daiane Boff ◽  
Karen Yu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Chemotactic Activity ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Agonist Activity ◽  
N Terminus ◽  
G Protein Coupled

The inflammatory human chemokine CXCL5 interacts with the G protein–coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg9 to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1–78), truncated CXCL5 [CXCL5(9–78)], and the citrullinated (Cit9) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and β-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca2+ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca2+ signaling assays with primary human CD14+ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

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.

scholarly journals Intersection of two key signal integrators in the cell: activator of G-protein signaling 3 and dishevelled-2

2020 ◽  
Vol 133 (17) ◽  
pp. jcs247908
Author(s):  
Ali Vural ◽  
Stephen M. Lanier
Keyword(s):  
Cell Surface ◽  
Signaling Pathways ◽  
G Protein ◽  
Functional Integration ◽  
Functional System ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Signaling Systems ◽  
A Cell ◽  
G Protein Coupled

ABSTRACTActivator of G-protein signaling 3 (AGS3, encoded by GPSM1) was discovered as a one of several receptor-independent activators of G-protein signaling, which are postulated to provide a platform for divergence between canonical and noncanonical G-protein signaling pathways. Similarly, Dishevelled (DVL) proteins serve as a point of divergence for β-catenin-dependent and -independent signaling pathways involving the family of Frizzled (FZD) ligands and cell-surface WNT receptors. We recently discovered the apparent regulated localization of dishevelled-2 (DVL2) and AGS3 to distinct cellular puncta, suggesting that the two proteins interact as part of various cell signaling systems. To address this hypothesis, we asked the following questions: (1) do AGS3 signaling pathways influence the activation of β-catenin (CTNNB1)-regulated transcription through the WNT–Frizzled–Dishevelled axis, and (2) is the AGS3 and DVL2 interaction regulated? The interaction of AGS3 and DVL2 was regulated by protein phosphorylation, subcellular distribution, and a cell-surface G-protein-coupled receptor. These data, and the commonality of functional system impacts observed for AGS3 and DVL2, suggest that the AGS3–DVL2 complex presents an unexpected path for functional integration within the cell.This article has an associated First Person interview with the first author of the paper.

scholarly journals Mechanisms of adhesion G protein–coupled receptor activation

2020 ◽  
Vol 295 (41) ◽  
pp. 14065-14083 ◽  
Author(s):  
Alexander Vizurraga ◽  
Rashmi Adhikari ◽  
Jennifer Yeung ◽  
Maiya Yu ◽  
Gregory G. Tall
Keyword(s):  
G Protein ◽  
Receptor Activation ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Physiological Processes ◽  
Class B ◽  
Peptide Agonist ◽  
G Protein Coupled ◽  
Existing Structure

Adhesion G protein–coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein–coupling seven-transmembrane–spanning bundle. GAIN domain–mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.

scholarly journals Purinergic Receptors Crosstalk with CCR5 to Amplify Ca2+ Signaling

2020 ◽  
Author(s):  
Mizuho Horioka ◽  
Emilie Ceraudo ◽  
Emily Lorenzen ◽  
Thomas P. Sakmar ◽  
Thomas Huber
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Purinergic Receptors ◽  
P2y Receptors ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Extracellular Milieu ◽  
Immunodeficiency Virus ◽  
Intracellular Ca ◽  
G Protein Coupled

AbstractMany G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C–C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce Ca2+ signaling. We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.

Engineered GPCRs as Tools to Modulate Signal Transduction

Physiology ◽  
2008 ◽  
Vol 23 (6) ◽  
pp. 313-321 ◽  
Author(s):  
Ying Pei ◽  
Sarah C. Rogan ◽  
Feng Yan ◽  
Bryan L. Roth
Keyword(s):  
Signal Transduction ◽  
Signaling Pathways ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Designer Drugs ◽  
Modulate Signal ◽  
Synthetic Drug ◽  
G Protein Coupled

Different families of G-protein-coupled receptors (GPCRs) have been engineered to provide exclusive control over the activation of these receptors and thus to understand better the consequences of their signaling in vitro and in vivo. These engineered receptors, named RASSLs (receptors activated solely by synthetic ligands) and DREADDs (designer receptors exclusively activated by designer drugs), are insensitive to their endogenous ligands but can be activated by synthetic drug-like compounds. Currently, the existing RASSLs and DREADDs cover the Gi, Gq, and Gs signaling pathways. These modified GPCRs can be utilized as ideal tools to study GPCR functions selectively in specific cellular populations.

The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4739-4747 ◽  
Author(s):  
Thalia Papayannopoulou ◽  
Gregory V. Priestley ◽  
Halvard Bonig ◽  
Betty Nakamoto
Keyword(s):  
G Protein ◽  
Underlying Mechanism ◽  
Progressive Increase ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Hematopoietic Stem ◽  
Colony Forming Unit

AbstractThe directed migration of mature leukocytes to inflammatory sites and the lymphocyte trafficking in vivo are dependent on G protein–coupled receptors and delivered through pertussis toxin (Ptx)–sensitive Gi-protein signaling. In the present study, we explored the in vivo role of G-protein signaling on the redistribution or mobilization of hematopoietic stem/progenitor cells (HPCs). A single injection of Ptx in mice elicits a long-lasting leukocytosis and a progressive increase in circulating colony-forming unit-culture (CFU-C) and colony-forming unit spleen (CFU-S). We found that the prolonged effect is sustained by a continuous slow release of Ptx bound to red blood cells or other cells and is potentially enhanced by an indirect influence on cell proliferation. Plasma levels of certain cytokines (interleukin 6 [IL-6], granulocyte colony-stimulating factor [G-CSF]) increase days after Ptx treatment, but these are unlikely initiators of mobilization. In addition to normal mice, mice genetically deficient in monocyte chemotactic protein 1 (MCP-1), matrix metalloproteinase 9 (MMP-9), G-CSF receptor, β2 integrins, or selectins responded to Ptx treatment, suggesting independence of Ptx-response from the expression of these molecules. Combined treatments of Ptx with anti–very late activation antigen (anti-VLA-4), uncovered potentially important insight in the interplay of chemokines/integrins, and the synergy of Ptx with G-CSF appeared to be dependent on MMP-9. As Ptx-mobilized kit+ cells display virtually no response to stromal-derived factor 1 (SDF-1) in vitro, our data suggest that disruption of CXCR4/SDF-1 signaling may be the underlying mechanism of Ptx-induced mobilization and indirectly reinforce the notion that active signaling through this pathway is required for continuous retention of cells within the bone marrow. Collectively, our data unveil a novel example of mobilization through pharmacologic modulation of signaling.

scholarly journals A Physiologically Required G Protein-coupled Receptor (GPCR)-Regulator of G Protein Signaling (RGS) Interaction That Compartmentalizes RGS Activity

2013 ◽  
Vol 288 (38) ◽  
pp. 27327-27342 ◽  
Author(s):  
Wayne Croft ◽  
Claire Hill ◽  
Eilish McCann ◽  
Michael Bond ◽  
Manuel Esparza-Franco ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Signaling ◽  
Rgs Proteins ◽  
Protein Signaling ◽  
Gpcr Signaling ◽  
Additional Layer ◽  
Physiological Relevance ◽  
Gtpase Cycle ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.

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