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.
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.
Group II activators of G-protein signaling play diverse functional roles through their interaction with G?i, G?t, and G?o via a G-protein regulatory (GPR) motif that serves as a docking site for G?-GDP. We recently reported the regulation of the AGS3-G?i signaling module by a cell surface, seven-transmembrane receptor. Upon receptor activation, AGS3 reversibly dissociates from the cell cortex, suggesting that it may function as a signal transducer with downstream signaling implications, and this question is addressed in the current report. In HEK-293 and COS-7 cells expressing the ?2A/D-AR and G?i3, receptor activation resulted in the translocation of endogenous AGS3 and AGS3-GFP from the cell cortex to a juxtanuclear region, where it co-localized with markers of the Golgi apparatus (GA). The agonist-induced translocation of AGS3 was reversed by the ?2-AR antagonist rauwolscine. The TPR domain of AGS3 was required for agonist-induced translocation of AGS3 from the cell cortex to the GA, and the translocation was blocked by pertussis toxin pretreatment or by the phospholipase C? inhibitor U73122. Agonist-induced translocation of AGS3 to the GA altered the functional organization and protein sorting at the trans-Golgi network. The regulated movement of AGS3 between the cell cortex and the GA offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the trans-Golgi network.
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.
Receptor-independent G-protein regulators provide diverse mechanisms for signal input to G-protein-based signaling systems, revealing unexpected functional roles for G-proteins. As part of a broader effort to identify disease-specific regulators for heterotrimeric G-proteins, we screened for such proteins in cardiac hypertrophy using a yeast-based functional screen of mammalian cDNAs as a discovery platform. We report the identification of three transcription factors belonging to the same family, transcription factor E3 (TFE3), microphthalmia-associated transcription factor, and transcription factor EB, as novel receptor-independent activators of G-protein signaling selective for G?(16). TFE3 and G?(16) were both up-regulated in cardiac hypertrophy initiated by transverse aortic constriction. In protein interaction studies in vitro, TFE3 formed a complex with G?(16) but not with G?(i3) or G?(s). Although increased expression of TFE3 in heterologous systems had no influence on receptor-mediated G?(16) signaling at the plasma membrane, TFE3 actually translocated G?(16) to the nucleus, leading to the induction of claudin 14 expression, a key component of membrane structure in cardiomyocytes. The induction of claudin 14 was dependent on both the accumulation and activation of G?(16) by TFE3 in the nucleus. These findings indicate that TFE3 and G?(16) are up-regulated under pathologic conditions and are involved in a novel mechanism of transcriptional regulation via the relocalization and activation of G?(16).
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.
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.
Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of G betagamma subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a G betagamma-dependent manner by providing a scaffold composed of G betagamma and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the G betagamma inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-G betagamma signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM.
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.
The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by ?60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.
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