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 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.

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 Light control of G protein signaling pathways by a novel photopigment

PLoS ONE ◽  
2018 ◽  
Vol 13 (10) ◽  
pp. e0205015
Author(s):  
Tomás Osorno ◽  
Oscar Arenas ◽  
Nelson J. Ramírez-Suarez ◽  
Fabio A. Echeverry ◽  
María del Pilar Gomez ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Light Control

scholarly journals Heterotrimeric G-Protein Signalers and RGSs in Aspergillus fumigatus

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 902
Author(s):  
Hee-Soo Park ◽  
Min-Ju Kim ◽  
Jae-Hyuk Yu ◽  
Kwang-Soo Shin
Keyword(s):  
Aspergillus Fumigatus ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
G Protein ◽  
G Proteins ◽  
Pathogenic Fungus ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Heterotrimeric G Protein ◽  
External Signals

The heterotrimeric G-protein (G-protein) signaling pathway is one of the most important signaling pathways that transmit external signals into the inside of the cell, triggering appropriate biological responses. The external signals are sensed by various G-protein-coupled receptors (GPCRs) and transmitted into G-proteins consisting of the α, β, and γ subunits. Regulators of G-protein signaling (RGSs) are the key controllers of G-protein signaling pathways. GPCRs, G-proteins, and RGSs are the primary upstream components of the G-protein signaling pathway, and they are highly conserved in most filamentous fungi, playing diverse roles in biological processes. Recent studies characterized the G-protein signaling components in the opportunistic pathogenic fungus Aspergillus fumigatus. In this review, we have summarized the characteristics and functions of GPCRs, G-proteins, and RGSs, and their regulatory roles in governing fungal growth, asexual development, germination, stress tolerance, and virulence in A. fumigatus.

scholarly journals Regulator of G-Protein Signaling 5 Reduces HeyA8 Ovarian Cancer Cell Proliferation and Extends Survival in a Murine Tumor Model

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Molly K. Altman ◽  
Duy T. Nguyen ◽  
Santosh B. Patel ◽  
Jada M. Fambrough ◽  
Aaron M. Beedle ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
G Protein ◽  
Expression System ◽  
Tumor Biology ◽  
Pathological Examination ◽  
Ovarian Cancer Cells ◽  
G Protein Signaling ◽  
Protein Signaling

The regulator of G-protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiated by extracellular mediators and G-protein-coupled receptors. RGS5 has an interesting dual biological role. One functional RGS5 role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other functional role is to promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumor progression—whether it plays a pathogenic or protective role in ovarian tumor biology. We thus constructed an inducible gene expression system to achieve RGS5 expression in HeyA8-MDR ovarian cancer cells. Through this we observed that inducible RGS5 expression significantly reducesin vitroBrdU-positive HeyA8-MDR cells, although this did not correlate with a reduction in tumor volume observed using anin vivomouse model of ovarian cancer. Interestingly, mice bearing RGS5-expressing tumors demonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observed by pathological examination. Additionally, mice bearing RGS5-expressing tumors were less likely to have ulcerated tumors. Taken together, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the context of a multicomponent approach for modulating tumor progression.

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