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.     

Identification and characterization of a G-protein regulatory motif in WAVE1

The G-protein regulatory (GPR) motif is a approximately 25 amino acid sequence that stabilizes the GDP-bound conformation of Gialpha. To identify additional GPR motifs, we expanded a motif-based search strategy and identified an additional 4 mammalian proteins (WAVE1-3, rat GHRH) and 10 plant proteins with candidate GPR motifs. The WAVE1 GPR peptide inhibited GTPgammaS binding to purified G-protein. Endogenous Gialpha and WAVE1 coimmunoprecipitated from brain lysates. A WAVE1-G-protein complex was also observed following transfection of COS7 cells with Gialpha3 and WAVE1. The docking of Gialpha within a WAVE1 scaffolding complex may facilitate dynamic cycling and/or targeting for efficient and localized control of actin polymerization.     

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.     

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.     

Inhibition of GDP/GTP exchange on G alpha subunits by proteins containing G-protein regulatory motifs

A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP > Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo.     

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.     

Identification of structural features in the G-protein regulatory motif required for regulation of heterotrimeric G-proteins.

The G-protein regulatory (GPR) motif, a conserved 25-30 amino acid domain found in multiple mammalian proteins, stabilizes the GDP-bound conformation of Galpha(i), inhibits guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Galpha(i) and competes for Gbetagamma binding to Galpha. To define the core GPR motif and key amino acid residues within a GPR peptide (TMGEEDFFDLLAKSQSKRMDDQRVDLAG), we determined the effect of truncation, insertion, and alanine substitutions on peptide-mediated inhibition of GTPgammaS binding to purified Galpha(i1). The bioactive core GPR peptide consists of 17 amino acids ((7)F-R(23)). Within this core motif, two hydrophobic sectors ((7)FF(8) and (10)LL(11)) and Q(22) are required for bioactivity, whereas M19A and R23A increased IC(50) values by 70-fold. Disruption of spatial relationships between the required sectors in the amino and carboxyl regions of the peptide also resulted in a loss of biological activity. Mutation of three charged sectors ((4)EED(6), R(18), (20)DD(21)) within the 28-amino acid GPR decreased peptide affinity by approximately 10-fold. Alanine substitutions of selected residues within the core GPR peptide differently influenced peptide inhibition of GTPgammaS binding to Galpha(i) versus Galpha(o). These data provide a platform for the development of novel, G-protein-selective therapeutics that inhibit Galpha(i)- mediated signaling, selectively activate Gbetagamma-sensitive effectors, and/or disrupt specific regulatory input to G-proteins mediated by GPR-containing proteins.     

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.     

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.     

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.     

A peptide core motif for binding to heterotrimeric G protein alpha subunits

Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.     

Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking

Chronic cocaine administration reduces G protein signaling efficacy. Here, we report that the expression of AGS3, which binds to GialphaGDP and inhibits GDP dissociation, was upregulated in the prefrontal cortex (PFC) during late withdrawal from repeated cocaine administration. Increased AGS3 was mimicked in the PFC of drug-naive rats by microinjecting a peptide containing the Gialpha binding domain (GPR) of AGS3 fused to the cell permeability domain of HIV-Tat. Infusion of Tat-GPR mimicked the phenotype of chronic cocaine-treated rats by manifesting sensitized locomotor behavior and drug seeking and by increasing glutamate transmission in nucleus accumbens. By preventing cocaine withdrawal-induced AGS3 expression with antisense oligonucleotides, signaling through Gialpha was normalized, and both cocaine-induced relapse to drug seeking and locomotor sensitization were prevented. When antisense oligonucleotide infusion was discontinued, drug seeking and sensitization were restored. It is proposed that AGS3 gates the expression of cocaine-induced plasticity by regulating G protein signaling in the PFC.     

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.     

Development of inhibitors of heterotrimeric Gαi subunits

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gα_i by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α₂-adrenoceptor (α₂AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gα_i. Molecular docking was used to identify potential molecules that bind to and stabilize the Gα_i–GDP complex by directly interacting with both Gα_i and GDP. Gα_i–GTP and Gα_q–GDP were used as a computational counter screen and Gα_q–GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gα_i. Several compounds showed Gα_i specific inhibition and were able to block α₂AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.     

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.     

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.     

The G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and Gialpha3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells

We addressed the role of the G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and G-proteins (Gialpha3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Gialpha3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Gialpha3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Gialpha. The influence of LGN and Gialpha3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry.     

In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display

The G protein regulatory (GPR) motif is a approximately 20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G(i/o)(alpha) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G(i)(alpha)(1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(i)(alpha)(1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G(i)(alpha)(1), inhibiting both the exchange of GDP in GTPgammaS binding assays and the AlF(4)(-)-stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G(betagamma) subunits for binding to G(i)(alpha)(1), suggesting their use as activators of G(betagamma) signaling.     

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.     

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.