Properties of biomolecular condensates defined by Activator of G-protein Signaling 3.

Activator of G-protein signaling 3 (AGS3; also known as GPSM1), a receptor-independent activator of G-protein signaling, oscillates among defined subcellular compartments and biomolecular condensates (BMCs) in a regulated manner that is likely related to the functional diversity of the protein. We determined the influence of cell stress on the cellular distribution of AGS3 and core material properties of AGS3 BMCs. Cellular stress (oxidative, pHi and thermal) induced the formation of AGS3 BMCs in HeLa and COS-7 cells, as determined by fluorescent microscopy. Oxidative stress-induced AGS3 BMCs were distinct from G3BP1 stress granules and from RNA processing BMCs defined by the P-body protein Dcp1a. Immunoblots indicated that cellular stress shifted AGS3, but not the stress granule protein G3BP1 to a membrane pellet fraction following cell lysis. The stress-induced generation of AGS3 BMCs was reduced by co-expression of the signaling protein Gαi3, but not the AGS3-binding partner DVL2. Fluorescent recovery following photobleaching of individual AGS3 BMCs indicated that there are distinct diffusion kinetics and restricted fluidity for AGS3 BMCs. These data suggest that AGS3 BMCs represent a distinct class of stress granules that serve as a previously unrecognized signal processing node.

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

Activator 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 ß-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 ß-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.

Role of G-proteins and phosphorylation in the distribution of AGS3 to cell puncta.

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) exhibits broad functional diversity and oscillates among different subcellular compartments in a regulated manner. AGS3 consists of a tetratricopeptide repeat (TPR) domain and a G-protein regulatory (GPR) domain. Here, we tested the hypothesis that phosphorylation of the AGS3 GPR domain regulates its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of wild-type (WT) AGS3, an AGS3 construct lacking all 24 potential phosphorylation sites in the GPR domain localized to cytosolic puncta. This change in localization was revealed to be dependent upon phosphorylation of a single threonine amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gαi and Gαo but not Gαs or Gαq Following treatment with alkaline phosphatase, both AGS3-T602A and WT AGS3 exhibited a gel shift in SDS-PAGE as compared to untreated WT AGS3, consistent with a loss of protein phosphorylation. The punctate distribution of AGS3-T602A was lost in an AGS3-A602T conversion mutant, but was still present upon T602 mutation to glutamate or aspartate. These results implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.

Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex.

Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gßγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαßγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oßγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gßγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oßγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαßγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.

Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

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 exhibit broad functionality and define a distinct core signaling triad.

Activators of G protein signaling (AGS), initially discovered in the search for receptor-independent activators of G protein signaling, define a broad panel of biologic 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 Gα and Gßγ independently of the classic heterotrimeric Gαßγ. 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; and group III, entities that bind to Gßγ. Group I AGS proteins can 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 Gα16 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.

Translocation of activator of G-protein signaling 3 to the Golgi apparatus in response to receptor activation and its effect on the trans-Golgi network.

Group II activators of G-protein signaling play diverse functional roles through their interaction with Gαi, Gαt, and Gαo via a G-protein regulatory (GPR) motif that serves as a docking site for Gα-GDP. We recently reported the regulation of the AGS3-Gαi 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 α2A/D-AR and Gαi3, 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 α2-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 Cß 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.

Group II activators of G-protein signaling: monitoring the interaction of Gα with the G-protein regulatory motif in the intact cell.

The G-protein regulatory (GPR) motif serves as a docking site for Gαi-GDP free of Gßγ. The GPR-Gα 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 Gα 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 Gαi and other binding partners in the cell. As an initial approach to this question, we established a platform to monitor the GPR-Gαi complex in intact cells using bioluminescence resonance energy transfer.

Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors.

Group II activators of G-protein signaling (AGS) serve as binding partners for Gα(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-Gα 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 Gα(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-Gα(i1)-YFP BRET were regulated by Ric-8A but not by Gα-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and Gα(i1)-YFP. The inhibitory regulation of GPR-Gα(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-Gα(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-Gα(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-G-Gα(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-Gα(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore Gα 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, Gα(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.

Influence of the accessory protein SET on M3 muscarinic receptor phosphorylation and G protein coupling.

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.

Identification of transcription factor E3 (TFE3) as a receptor-independent activator of Gα16: gene regulation by nuclear Gα subunit and its activator.

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 Gα(16). TFE3 and Gα(16) were both up-regulated in cardiac hypertrophy initiated by transverse aortic constriction. In protein interaction studies in vitro, TFE3 formed a complex with Gα(16) but not with Gα(i3) or Gα(s). Although increased expression of TFE3 in heterologous systems had no influence on receptor-mediated Gα(16) signaling at the plasma membrane, TFE3 actually translocated Gα(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 Gα(16) by TFE3 in the nucleus. These findings indicate that TFE3 and Gα(16) are up-regulated under pathologic conditions and are involved in a novel mechanism of transcriptional regulation via the relocalization and activation of Gα(16).

Loss of activator of G-protein signaling 3 impairs renal tubular regeneration following acute kidney injury in rodents.

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 Gßγ subunit activity, and lentiviral overexpression of the carboxyl-terminus of G-protein-coupled receptor kinase 2 (GRK2ct), a scavenger of Gßγ 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.

Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gα(olf) for the first time and unprecedented yield of Gα(q) and Gα(13). Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gα(olf) showed that it binds GTPγS at a rate marginally slower than Gα(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gα(s short).

Regulation of the AGS3·G{alpha}i signaling complex by a seven-transmembrane span receptor.

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 Gα(i) free of Gßγ 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·Gα(i) signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα(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 α(2)-adrenergic receptors or µ-opioid receptors reduced AGS3-RLuc·Gα(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 Gßγ sequestration with the carboxyl terminus of GRK2. Gα(i)-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα(i) and/or Gα(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 Gαßγ and GPR-Gα(i) offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Activator of G protein signaling 3 promotes epithelial cell proliferation in PKD.

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.

Distribution of activator of G-protein signaling 3 within the aggresomal pathway: role of specific residues in the tetratricopeptide repeat domain and differential regulation by the AGS3 binding partners Gi(alpha) and mammalian inscuteable.

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.

Activator of G protein signaling 8 (AGS8) is required for hypoxia-induced apoptosis of cardiomyocytes: role of G betagamma and connexin 43 (CX43).

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.

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

Activator of G protein signaling 3 null mice: I. Unexpected alterations in metabolic and cardiovascular function.

Activator of G protein signaling (AGS)-3 plays functional roles in cell division, synaptic plasticity, addictive behavior, and neuronal development. As part of a broad effort to define the extent of functional diversity of AGS3-regulated-events in vivo, we generated AGS3 null mice. Surprisingly, AGS3 null adult mice exhibited unexpected alterations in cardiovascular and metabolic functions without any obvious changes in motor skills, basic behavioral traits, and brain morphology. AGS3 null mice exhibited a lean phenotype, reduced fat mass, and increased nocturnal energy expenditure. AGS3 null mice also exhibited altered blood pressure control mechanisms. These studies expand the functional repertoire for AGS3 and other G protein regulatory proteins providing unexpected mechanisms by which G protein systems may be targeted to influence obesity and cardiovascular function.

Signaling by a non-dissociated complex of G protein ßγ and α subunits stimulated by a receptor-independent activator of G protein signaling, AGS8.

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gbetagamma subunits. Results are presented demonstrating that AGS8 binds to the effector and alpha subunit binding "hot spot" on Gbetagamma yet does not interfere with Galpha subunit binding to Gbetagamma or phospholipase C beta2 activation. AGS8 stimulates activation of phospholipase C beta2 by heterotrimeric Galphabetagamma and forms a quaternary complex with Galpha(i1), Gbeta(1)gamma(2), and phospholipase C beta2. AGS8 rescued phospholipase C beta binding and regulation by an inactive beta subunit with a mutation in the hot spot (beta(1)(W99A)gamma(2)) that normally prevents binding and activation of phospholipase C beta2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gbetagamma interactions with phospholipase C beta2. Mutation of an alternate binding site for phospholipase C beta2 in the amino-terminal coiled-coil region of Gbetagamma prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gbetagamma binding proteins, for directing Gbetagamma signaling through alternative effector activation sites on Gbetagamma in the absence of subunit dissociation.