Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha

Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.     

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 Galpha(i). 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 alpha(2)-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galpha(i3) in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galpha(i3) indicating that the interaction of AGS3 with Frmpd1 and Galpha(i3) is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galpha(i) in a regulated manner.     

Receptor-independent activators of heterotrimeric G-protein signaling pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.     

Activator of G-protein signaling in asymmetric cell divisions of the sea urchin embryo

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.     

Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins

AGS3 (activator of G-protein signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. As an initial approach to define the role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of G-protein. Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT(1)-MF2. Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with Galpha(i3) from cell and tissue lysates, indicating that a subpopulation of AGS3 and Galpha(i) exist as a complex in the cell. The coimmunoprecipitation of AGS3 and Galpha(i) was dependent upon the conformation of Galpha(i3) (GDP GTPgammaS (guanosine 5'-3-O-(thio)triphosphate)). The regions of AGS3 that bound Galpha(i) were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro(463)-Ser(650)), each of which were capable of binding Galpha(i). AGS3-GPR domains selectively interacted with Galpha(i) in tissue and cell lysates and with purified Galpha(i)/Galpha(t). Subsequent experiments with purified Galpha(i2) and Galpha(i3) indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one Galpha(i) subunit at the same time. The AGS3-GPR domains effectively competed with Gbetagamma for binding to Galpha(t(GDP)) and blocked GTPgammaS binding to Galpha(i1). AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.     

Expression analysis and subcellular distribution of the two G-protein regulators AGS3 and LGN indicate distinct functionality. Localization of LGN to the midbody during cytokinesis

Activator of G-protein signaling 3 (AGS3) and LGN have a similar domain structure and contain four G-protein regulatory motifs that serve as anchors for the binding of the GDP-bound conformation of specific G-protein alpha subunits. As an initial approach to define further the different functional roles of AGS3 and LGN, we determined their expression profile and subcellular distribution. AGS3- and LGN-specific antisera indicated a widespread tissue distribution of LGN, whereas AGS3 is primarily enriched in brain. Brain punch biopsies of 13 discrete brain regions indicated that both AGS3 and LGN are expressed in all areas tested but are differentially regulated during development. LGN is expressed in neuronal, astroglial, and microglial cultures, whereas AGS3 expression is restricted to neurons. In primary neuronal cultures as well as in dividing cultures of PC12 cells, immunocytochemistry indicated distinct subcellular locations of AGS3 and LGN. The subcellular locations of the two proteins were differentially regulated by external stimuli and the cell cycle. In PC12 and COS7 cells, LGN moves from the nucleus to the midbody structure separating daughter cells during the later stages of mitosis, suggesting a role for G-proteins in cytokinesis. Thus, although AGS3 and LGN share a similar overall motif structure and both bind G-proteins, nature has endowed these proteins with different regulatory elements that allow functional diversity by virtue of tissue-specific expression and subcellular positioning.     

AGS3 inhibits GDP dissociation from galpha subunits of the Gi family and rhodopsin-dependent activation of transducin

A number of recently discovered proteins that interact with the alpha subunits of G(i)-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs. Activator of G protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats. To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by G(i)alpha(1) and transducin-alpha (G(t)alpha). The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to G(i)alpha and rhodopsin-stimulated GTPgammaS binding to G(t)alpha. The full-length AGS3 GPR domain, AGS3-(463-650), was approximately 30-fold more potent than AGS3-(572-629), containing two AGS3 GPR motifs. The IC(50) values for the AGS3-(463-650) inhibitory effects on G(i)alpha and transducin were 0.12 and 0.15 microm, respectively. Furthermore, AGS3-(463-650) and AGS3-(572-629) effectively blocked the GDP release from G(i)alpha and rhodopsin-induced dissociation of GDP from G(t)alpha. The potencies of AGS3-(572-629) and AGS3-(463-650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTPgammaS binding. Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by G(i)alpha. The catalytic rate of G(t)alpha GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463-650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of Gbetagamma signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor.     

AGS3 and signal integration by Galpha(s)- and Galpha(i)-coupled receptors: AGS3 blocks the sensitization of adenylyl cyclase following prolonged stimulation of a Galpha(i)-coupled receptor by influencing processing of Galpha(i)

AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDP-bound conformation of Galpha(i) > Galpha(o). AGS3 and related proteins may influence signal strength or duration as well as the adaptation of the signaling system associated with sustained stimulation. To address these issues, we determined the effect of AGS3 on the integration of stimulatory (Galpha(s)-mediated vasoactive intestinal peptide receptor) and inhibitory (Galpha(i)-mediated alpha(2)-adrenergic receptor (alpha(2)-AR)) signals to adenylyl cyclase in Chinese hamster ovary cells. AGS3-SHORT and AGS3-LONG did not alter the VIP-induced increase in cAMP or the inhibitory effect of alpha(2)-AR activation. System adaptation was addressed by determining the influence of AGS3 on the sensitization of adenylyl cyclase that occurs following prolonged activation of a Galpha(i)-coupled receptor. Incubation of cells with the alpha(2)-AR agonist UK14304 (1 microm) for 18 h resulted in a approximately 1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated Galpha(i3). Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of Galpha(i3) decay. A mutant AGS3-SHORT incapable of binding G-protein was inactive. These data suggest that AGS3 and perhaps other G-protein regulatory motif-containing proteins increase the stability of Galpha(i) in the membrane, which influences the adaptation of the cell to prolonged activation of Galpha(i)-coupled receptors.     

Influence of cytosolic AGS3 on receptor--G protein coupling

Activator of G protein signaling 3 (AGS3) activates the Gbetagamma mating pathway in yeast in a manner that is independent of heptahelical receptors. It competes with Gbetagamma subunits to bind GDP-bound Gi/o(alpha) subunits via four repeated G protein regulatory (GPR) domains in the carboxyl-terminal half of the molecule. However, little is known about the functional role of AGS3 in cellular signaling. Here the effect of AGS3 on receptor-G protein coupling was examined in an Sf9 cell membrane-based reconstitution system. A GST-AGS3-GPR fusion protein containing the four individual AGS3-GPR domains inhibits receptor coupling to Galpha subunits as effectively as native AGS3 and more effectively than GST fusion proteins containing the individual AGS3-GPR domains. While none of the GPR domains distinguished among the three G(i)alpha subunits, both individual and full-length GPR domains interacted more weakly with G(o)alpha than with G(i)alpha. Cytosolic AGS3, but not membrane-associated AGS3, can interact with G(i)alpha subunits and disrupt their receptor coupling. Immunoblotting studies reveal that cytosolic AGS3 can remove G(i)alpha subunits from the membrane and sequester G(i)alpha subunits in the cytosol. These findings suggest that AGS3 may downregulate heterotrimeric G protein signaling by interfering with receptor coupling.     

Thermodynamic characterization of the binding of activator of G protein signaling 3 (AGS3) and peptides derived from AGS3 with G alpha i1

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G alpha i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G alpha i1. The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G alpha i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (Kd approximately 20 nm) and two apparent low affinity (Kd approximately 300 nm) binding sites for G alpha i1. Upon deletion of the C-terminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (Kd approximately 400 nm) for G alpha i1. Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to G alpha i1 with Kd values in the range of 1-8 microm. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5'-3-O-(thio)triphosphate to G alpha i1, GPR3 did not. However, addition of N- and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G alpha i1 and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward G alpha i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.     

Receptor-independent activators of heterotrimeric G-proteins

Heterotrimeric G-protein signalling systems are primarily activated via cell surface receptors possessing the seven membrane span motif. Several observations suggest the existence of other modes of input to such signalling systems either downstream of effectors or at the level of G-proteins themselves. Using a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae, we identified three proteins, AGS1-3 (for Activators of G-protein Signalling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1 defines a distinct member of the super family of ras related proteins. AGS2 is identical to mouse Tctex1, a protein that exists as a light chain component of the cytoplasmic motor protein dynein and subserves as yet undefined functions in cell signalling pathways. AGS3 possesses a series of tetratrico repeat motifs and a series of four amino acid repeats termed G-protein regulatory motifs. The GPR motifs are found in a number of proteins that interact with and regulate Galpha. Although each AGS protein activates G-protein signaling, they do so by different mechanisms within the context of the G-protein activation/deactivation cycle. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signalling pathways.     

Regulation of the AGS3·Gαi 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 Gβγ 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 α₂-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 Gβγ 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 Gαβγ and GPR-Gα_i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.     

Activator of G-protein signaling 1 blocks GIRK channel activation by a G-protein-coupled receptor: apparent disruption of receptor signaling complexes

The Ras-related protein, activator of G-protein signaling 1 (AGS1) or Dexras1, interacts with G(i)/G(o)alpha and activates heterotrimeric G-protein signaling systems independent of a G-protein-coupled receptor (GPCR). As an initial approach to further define the cellular role of AGS1 in GPCR signaling, we determined the influence of AGS1 on the regulation of G(betagamma)-regulated inwardly rectifying K(+) channel (GIRK) current (I(ACh)) by M(2)-muscarinic receptor (M(2)-MR) in Xenopus oocytes. AGS1 expression inhibited receptor-mediated current activation by >80%. Mutation of a key residue (G31V) within the G(1) domain involved in nucleotide binding for Ras-related proteins eliminated the action of AGS1. The inhibition of I(ACh) was not overcome by increasing concentrations of the muscarinic agonist acetylcholine but was progressively lost upon injection of increasing amounts of M(2)-MR cRNA. These data suggest that AGS1 may antagonize GPCR signaling by altering the pool of heterotrimeric G-proteins available for receptor coupling and/or disruption of a preformed signaling complex. Such regulation would be of particular importance for those receptors that exist precoupled to heterotrimeric G-protein and for receptors operating within signaling complexes.     

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 Giα 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 Giα-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 Giα and mammalian Inscuteable (mInsc). Giα 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.The discovery of AGS3 (GPSM1) and related accessory proteins revealed unexpected functional diversity for G-protein signaling systems (8, 36). AGS3 is involved in a number of different cellular activities, including asymmetric cell division during neuronal development (30), neuronal plasticity and addiction (9, 10, 12, 38, 39), autophagy (27), membrane protein trafficking (17), cardiovascular function (7), and metabolism (7). AGS3 is a multidomain protein consisting of seven tetratricopeptide repeats (TPR) in the amino-terminal portion of the protein and four G-protein regulatory (GPR) motifs in the carboxyl region of the protein. Each of the GPR motifs binds and stabilizes the GDP-bound conformation of Gα (Giα, Gtα, and Gi/oα), essentially behaving as a guanine nucleotide dissociation inhibitor. As such, AGS3 may be complexed with up to four Gα and function as an alternative binding partner for Gα independently of the classical heterotrimeric Gαβγ. Despite the clearly demonstrated function of AGS3 and the related protein LGN (GPSM2 or AGS5) in various model organisms and a fairly solid, basic biochemical understanding of the interaction of a GPR motif with Gα, the signals that operate “upstream” and/or “downstream” of AGS3 or an AGS3-Gi/oα complex are not well defined.AGS3 and other GPR proteins may regulate G-protein signaling directly by influencing the interaction of Gα with Gβγ or another Gα binding partner. In addition, a portion of Gα in the cell is complexed with GPR proteins to various degrees, and this interaction is regulated. Ric-8A interacts with an AGS3-Giα complex in a manner somewhat analogous to the interaction of a G-protein-coupled receptor with heterotrimeric Gαβγ, promoting nucleotide exchange and the apparent dissociation of AGS3 and Giα-GDP (37). The specific impact of AGS3 and other GPR proteins on signaling events is likely dependent upon where the individual protein is positioned within the cell and the nature of intra- and intermolecular interactions that influence the interaction of the GPR motif with Gi/oα.The TPR domain of AGS3 is an important determinant of its positioning within the cell through its interaction with specific binding partners (1, 8, 28, 36). As part of a broader effort to address the fundamental questions of AGS3 “positioning” and control of G-protein interaction, we focused upon the roles of individual TPR domains. Endogenous and ectopically expressed wild-type AGS3 is nonhomogeneously distributed in the cytoplasm, with obvious punctate structures, and it may be present at the cell periphery. Disruption of the TPR organizational structure by targeted amino acid substitutions or introduction of a nonsynonymous single-nucleotide polymorphism redistributes AGS3 to punctate structures throughout the cytoplasm that are similar in appearance to the preaggresomal assemblies or aggregates observed in neurodegenerative diseases. Upon cellular stress, both wild-type and TPR-modified AGS3 migrate, in a microtubule-dependent manner, to a perinuclear aggresome. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is differentially regulated by Giα and mammalian Inscuteable (mInsc), which bind to the GPR and TPR domains, respectively, of AGS3. These data present AGS3 and G-proteins as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies and suggest that this involvement can be manipulated to therapeutic advantage.     

Stabilization of the GDP-bound conformation of Gialpha by a peptide derived from the G-protein regulatory motif of AGS3

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this approximately 20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Gialpha1,2 in protein interaction assays, inhibited guanosine 5'-O-(3-thiotriphosphate) binding to Gialpha, and stabilized the GDP-bound conformation of Gialpha. The GPR peptide had little effect on nucleotide binding to Goalpha and brain G-protein indicating selective regulation of Gialpha. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gialpha. The GPR consensus peptide also blocked receptor coupling to Gialphabetagamma indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Gialpha, this conformation of Gialpha(GDP) was not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gialpha- and Gbetagamma-regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.     

Receptor- and nucleotide exchange-independent mechanisms for promoting G protein subunit dissociation

Mechanisms for heterotrimeric G protein activation that do not rely on G protein coupled receptor activation are becoming increasingly apparent. We recently identified beta gamma subunit-binding peptides that we proposed bound to a "hot spot" on beta gamma subunits, stimulating G protein dissociation without stimulating nucleotide exchange and activating G protein signaling in intact cells. AGS3, a member of the activators of G protein signaling family of proteins, also activates G protein signaling in a nucleotide exchange-independent manner, and AGS3 homologues are involved in asymmetric cell division during development. Here we demonstrate that a consensus G protein regulatory (GPR) peptide from AGS3 and related proteins is sufficient to induce G protein subunit dissociation and that both the GPR and hot spot-binding peptides promote dissociation to extents comparable with a known G protein activator, AMF. Peptides derived from adenylyl cyclase 2 and GRK2 prevented formation of the heterotrimeric complex but did not alter the rate of alpha subunit dissociation from beta gamma subunits. These data indicate that these nucleotide exchange-independent G protein activator peptides do not simply compete for alpha interactions with beta gamma subunits, but actively promote subunit dissociation. Thus, we propose two novel mechanisms for nucleotide exchange independent activation of G protein signaling, one that involves conformational changes in the alpha subunit and one that involves conformational changes in the beta gamma subunits.     

RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity

The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity.     

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration

Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Galpha(i)/Galpha(o) heterotrimeric signaling pathways. In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Galpha(i)/Galpha(o). AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.     

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.