Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.
To interrogate why redox homeostasis and glutathione S-transferase P (GSTP) are important in regulating bone marrow cell proliferation and migration, we isolated crude bone marrow, lineage negative and bone marrow derived-dendritic cells (BMDDCs) from both wild type (WT) and knockout (Gstp1/p2(-/-)) mice. Comparison of the two strains showed distinct thiol expression patterns. WT had higher baseline and reactive oxygen species-induced levels of S-glutathionylated proteins, some of which (sarco-endoplasmic reticulum Ca2(+)-ATPase) regulate Ca(2+) fluxes and subsequently influence proliferation and migration. Redox status is also a crucial determinant in the regulation of the chemokine system. CXCL12 chemotactic response was stronger in WT cells, with commensurate alterations in plasma membrane polarization/permeability and intracellular calcium fluxes; activities of the downstream kinases, ERK and Akt were also higher in WT. In addition, expression levels of the chemokine receptor CXCR4 and its associated phosphatase, SHP-2, were higher in WT. Inhibition of CXCR4 or SHP2 decreased the extent of CXCL12-induced migration in WT BMDDCs. The differential surface densities of CXCR4, SHP-2 and inositol trisphosphate receptor in WT and Gstp1/p2(-/-) cells correlated with the differential CXCR4 functional activities, as measured by the extent of chemokine-induced directional migration and differences in intracellular signaling. These observed differences contribute to our understanding of how genetic ablation of GSTP causes higher levels of myeloproliferation and migration.
The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by siRNA induced apoptosis in A2780ip2, OVCAR5, and ES-2 ovarian cancer cells, correlating with loss of the pro-survival protein Mcl-1 through a GSK-3?-independent mechanism. SiRNA to EDD or Mcl-1 induced comparable levels of apoptosis in A2780ip2 and ES-2 cells. Stable overexpression of Mcl-1 protected cells from apoptosis following EDD knockdown, accompanied by a loss of endogenous, but not exogenous, Mcl-1 protein, suggesting that EDD regulated Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 mRNA and EDD transfection enhanced murine Mcl-1 promoter driven luciferase expression 5-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and showed that these cells were four- to 24-fold more sensitive to cisplatin. Moreover, transient EDD overexpression in COS-7 cells was sufficient to promote cisplatin resistance 2.4-fold, dependent upon its E3 ligase activity. In vivo, mouse intraperitoneal ES-2 and A2780ip2 xenograft experiments showed that mice treated with EDD siRNA by nanoliposomal delivery (DOPC) and cisplatin had significantly less tumor burden than those treated with control siRNA/DOPC alone (ES-2, 77.9% reduction, p=0.004; A2780ip2, 75.9% reduction, p=0.042) or control siRNA/DOPC with cisplatin in ES-2 (64.4% reduction, p=0.035), with a trend in A2780ip2 (60.3% reduction, p=0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest EDD is a therapeutic target for ovarian cancer.
Activators of G-protein signaling (AGS), initially discovered in the search for receptor-independent activators of G-protein signaling, define a broad panel of biological regulators that influence signal transfer from receptor to G-protein, guanine nucleotide binding and hydrolysis, G-protein subunit interactions and/or serve as alternative binding partners for G? and G?? independent of the classical heterotrimeric G??. AGS proteins generally fall into three groups based upon their interaction with and regulation of G-protein subunits: Group I - guanine nucleotide exchange factors (GEF), Group II - guanine nucleotide dissociation inhibitors, Group III - bind to G??. Group I AGS proteins may engage all subclasses of G-proteins, whereas Group II AGS proteins primarily engage the Gi/Go/transducin family of G-proteins. A fourth group of AGS proteins, with selectivity for G?16 may be defined by the Mitf-Tfe family of transcription factors. Groups I-III may act in concert generating a core signaling triad analogous to the core triad for heterotrimeric G-proteins (GEF - G-proteins - Effector). These two core triads may function independently of each other or actually cross-integrate for additional signal processing. AGS proteins have broad functional roles and their discovery has advanced new concepts in signal processing, cell and tissue biology, receptor pharmacology and system adaptation providing unexpected platforms for therapeutic and diagnostic development.
The G-protein regulatory (GPR) motif serves as a docking site for G?i-GDP free of G??. The GPR-G? complex may function at the cell cortex and/or at intracellular sites. GPR proteins include the Group II Activators of G-protein signaling identified in a functional screen for receptor-independent activators of G-protein signaling (GPSM1-3, RGS12) each of which contain 1-4 GPR motifs. GPR motifs are also found in PCP2/L7(GPSM4), Rap1-Gap1 Transcript Variant 1, and RGS14. While the biochemistry of the interaction of GPR proteins with purified G? is generally understood, the dynamics of this signaling complex and its regulation within the cell remains undefined. Major questions in the field revolve around the factors that regulate the subcellular location of GPR proteins and their interaction with G?i and other binding partners in the cell. As an initial approach to this question, we established a platform to monitor the GPR-G?i complex in intact cells using bioluminescence resonance energy transfer.
In macrophages autophagy assists antigen presentation, affects cytokine release, and promotes intracellular pathogen elimination. In some cells autophagy is modulated by a signaling pathway that employs G?i3, Activator of G-protein Signaling-3 (AGS3/GPSM1), and Regulator of G-protein Signaling 19 (RGS19). As macrophages express each of these proteins, we tested their importance in regulating macrophage autophagy. We assessed LC3 processing and the formation of LC3 puncta in bone marrow derived macrophages prepared from wild type, Gnai3(-/-), Gpsm1(-/-), or Rgs19(-/-) mice following amino acid starvation or Nigericin treatment. In addition, we evaluated rapamycin-induced autophagic proteolysis rates by long-lived protein degradation assays and anti-autophagic action after rapamycin induction in wild type, Gnai3(-/-), and Gpsm1(-/-) macrophages. In similar assays we compared macrophages treated or not with pertussis toxin, an inhibitor of GPCR (G-protein couple receptor) triggered G?i nucleotide exchange. Despite previous findings, the level of basal autophagy, autophagic induction, autophagic flux, autophagic degradation and the anti-autophagic action in macrophages that lacked G?i3, AGS3, or RGS19; or had been treated with pertussis toxin, were similar to controls. These results indicate that while G?i signaling may impact autophagy in some cell types it does not in macrophages.
Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional G?(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds G?(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·G?(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a G?(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the ?(2A)-adrenergic receptor (?(2A)-AR) in a G?(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·?(2A)-AR complex occurs in the presence of G?(i/o) but not G?(s) or G?(q). Unexpectedly, RGS14 does not dissociate from G?(i1) in the presence of stimulated ?(2A)-AR, suggesting preservation of RGS14·G?(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·G?(i1) complex and the G?(i1)-dependent RGS14·?(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on G?(i/o) and that these RGS14·G?(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.
To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.
The intracellular mechanisms underlying renal tubular epithelial cell proliferation and tubular repair following ischemia-reperfusion injury (IRI) remain poorly understood. In this report, we demonstrate that activator of G-protein signaling 3 (AGS3), an unconventional receptor-independent regulator of heterotrimeric G-protein function, influences renal tubular regeneration following IRI. In rat kidneys exposed to IRI, there was a temporal induction in renal AGS3 protein expression that peaked 72 h after reperfusion and corresponded to the repair and recovery phase following ischemic injury. Renal AGS3 expression was localized predominantly to the recovering outer medullary proximal tubular cells and was highly coexpressed with Ki-67, a marker of cell proliferation. Kidneys from mice deficient in the expression of AGS3 exhibited impaired renal tubular recovery 7 d following IRI compared to wild-type AGS3-expressing mice. Mechanistically, genetic knockdown of endogenous AGS3 mRNA and protein in renal tubular epithelial cells reduced cell proliferation in vitro. Similar reductions in renal tubular epithelial cell proliferation were observed following incubation with gallein, a selective inhibitor of G?? subunit activity, and lentiviral overexpression of the carboxyl-terminus of G-protein-coupled receptor kinase 2 (GRK2ct), a scavenger of G?? subunits. In summary, these data suggest that AGS3 acts through a novel receptor-independent mechanism to facilitate renal tubular epithelial cell proliferation and renal tubular regeneration.
Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein ? subunits. Co-expression of G? subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted G? protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein ? subunit purification that was applicable to all G? subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein G?(olf) for the first time and unprecedented yield of G?(q) and G?(13). G? subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·G? complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. G? subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5-[?-thio]triphosphate (GTP?S), and stimulate appropriate G protein effector enzymes. A primary characterization of G?(olf) showed that it binds GTP?S at a rate marginally slower than G?(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G?(s short).
G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind G?(i) free of G?? providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·G?(i) signaling module by a cell surface, seven-transmembrane receptor. AGS3 and G?(i1) tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of ?(2)-adrenergic receptors or ?-opioid receptors reduced AGS3-RLuc·G?(i1)-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by G?? sequestration with the carboxyl terminus of GRK2. G?(i)-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to G?(i) and/or G?(o) indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both G??? and GPR-G?(i) offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.
The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA-induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 +/- 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA-expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Galpha subunit function and control epithelial cell function in PKD.
Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind Galpha(i)-GDP subunits free of Gbetagamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Galpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Galpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Galpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were Galpha(i)-dependent and reduced by agonist, indicating that AGS4-Galpha(i) complexes are receptor-proximal. These data suggest that AGS4-Galpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.
AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.
Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active G?(i/o)-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive G?(i1/3)-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive G?(i1)-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive G?(i1)-GDP but not active G?(i1)-GTP. Active H-Ras(G/V) interacts with a native RGS14·G?(i1) complex in brain lysates, and co-expression of RGS14 and G?(i1) in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the G?(i)-linked ?(2A)-adrenergic receptor induces a conformational change in the G?(i1)·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive G?(i1)-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.
Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1(V/V) mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1(+/+) and Gpsm1(+/-) mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via G?? subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease.
Group II activators of G-protein signaling (AGS) serve as binding partners for G?(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-G? signaling modules may be differentially regulated by cell surface receptors or by different nonreceptor guanine nucleotide exchange factors. We determined the effect of the nonreceptor guanine nucleotide exchange factors AGS1, GIV/Girdin, and Ric-8A on the interaction of two distinct GPR proteins, AGS3 and AGS4, with G?(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-G?(i1)-YFP and AGS4-Rluc-G?(i1)-YFP BRET were regulated by Ric-8A but not by G?-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and G?(i1)-YFP. The inhibitory regulation of GPR-G?(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-G?(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-G?(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-G?(i1)-YFP and AGS4-Rluc-G-G?(i1)-YFP BRET were observed in both pellet and supernatant subcellular fractions and were regulated by Ric-8A in both fractions. The regulation of the GPR-G?(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore G? expression in Ric8A(-/-) mouse embryonic stem cells, involved two helical domains at the carboxyl terminus of Ric-8A. These data indicate a dynamic interaction between GPR proteins, G?(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.
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