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.     

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.     

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.     

Ric-8A catalyzes guanine nucleotide exchange on G alphai1 bound to the GPR/GoLoco exchange inhibitor AGS3

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Galpha subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Galpha.GDP subunits and forms a stable nucleotide-free Ric-8A:Galpha complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Galpha subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Galpha.GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Galpha(il) and that of Ric-8A with the AGS3-C:Galpha(il).GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Galpha(i1).GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Galpha(i1).GDP. Subsequent dissociation of AGS3-C and GDP from Galpha(i1) yields a stable nucleotide free Ric-8A.Galpha(i1) complex that, in the presence of GTP, dissociates to yield Ric-8A and Galpha(i1).GTP. AGS3-C does not induce dissociation of the Ric-8A.Galpha(i1) complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Galpha(i1).GDP ensures unidirectional activation of Galpha subunits that cannot be reversed by AGS3.     

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.     

Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Galpha(i2,3)

The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [3H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (K(d) = 48.7 pm) and saturable (B(max) = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) inhibited [3H]DHEA binding to plasma membranes by 69%, and DHEA increased [35S]GTPgammaS binding by 157%. DHEA stimulated [35S]GTPgammaS binding to Galpha(i2) and Galpha(i3), but not to Galpha(i1) or Galpha(o). Pretreatment of plasma membranes with antibody to Galpha(i2) or Galpha(i3), but not to Galpha(i1), inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Galpha(i2) and Galpha(i3). These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.     

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.     

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.     

Functional coupling with Galpha(q) and Galpha(i1) protein subunits promotes high-affinity agonist binding to the neurotensin receptor NTS-1 expressed in Escherichia coli

To analyze the coupling of Galpha subunits to the rat neurotensin receptor NTS-1 (NTR), fusion proteins were expressed in Escherichia coli with various Galpha subunits covalently linked to the receptor C-terminus. The presence of Galpha(q) or Galpha(i/q), in which the six C-terminal residues of Galpha(i1) were replaced with those from Galpha(q), increased the percentage of receptors in the agonist high-affinity state. This effect was less pronounced for wild-type Galpha(i1) and not observed for Galpha(i/s). Functional coupling of neurotensin receptor to Galpha was demonstrated by neurotensin-induced [(35)S]GTPgammaS binding for the Galpha(q), Galpha(i/q) and Galpha(i1) subunits, but not for Galpha(i/s). Our results extend previous findings of the dual coupling of NTR to pertussis toxin-sensitive and -insensitive G-proteins in Chinese hamster ovary cells with preference for the latter.     

Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.     

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.     

Identification and characterization of AGS4: a protein containing three G-protein regulatory motifs that regulate the activation state of Gialpha

Activators of G-protein signaling 1-3 (AGS1-3) were identified in a functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor. We report the isolation and characterization of an additional AGS protein (AGS4) from a human prostate leiomyosarcoma cDNA library. AGS4 is identical to G18.1b, which is encoded by a gene within the major histocompatibility class III region of chromosome 6. The activity of AGS4 in the yeast-based functional screen was selective for G(i2)/G(i3) and independent of guanine-nucleotide exchange by G(i)alpha. RNA blots indicated enrichment of AGS4/G18.1b mRNA in heart, placenta, lung, and liver. Immunocytochemistry with AGS4/G18.1b-specific antisera indicated a predominant nonhomogeneous, extranuclear distribution within the cell following expression in COS7 or Chinese hamster ovary cells. AGS4/G18.1b contains three G-protein regulatory motifs downstream of an amino terminus domain with multiple prolines. Glutathione S-transferase (GST)-AGS4/G18.1b fusion proteins interacted with purified G(i)alpha, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to purified G(i)alpha(1). AGS4/G18.1b was also complexed with G(i)alpha(3) in COS7 cell lysates following cell transfection. However, AGS4/G18.1b did not alter the generation of inositol phosphates in COS7 cells cotransfected with the Gbetagamma-regulated effector phospholipase C-beta2. These data suggest either that an additional signal is required to position AGS4/G18.1b in the proper cellular location where it can access heterotrimer and promote subunit dissociation or that AGS4 serves as an alternative binding partner for G(i)alpha independent of Gbetagamma participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Galpha and Gbetagamma subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Galpha and Gbetagamma) following agonist-induced GPCR (alpha(2A)-adrenergic receptor; alpha(2A)-AR) activation in a cell-free assay system. alpha(2A)-AR membranes were reconstituted with the G-proteins (+/-hexahistidine-tagged) Galpha(i1) and Gbeta1gamma2 and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [35S]GTPgammaS. In the presence of Ni(2+)-coated agarose beads, the activated his-tagged Galpha(i1)his-[35S]GTPgammaS complex was captured on the Ni(2+)-presenting surface. When his-tagged Gbeta1gamma2 (Gbeta1gamma2his) was used with Galpha(i1), the [35S]GTPgammaS-bound Galpha(i1) was not present on the Ni(2+)-coated beads, but rather, it was separated from the beta1gamma2(his)-beads, demonstrating receptor-induced dissociation of Galpha and Gbetagamma subunits. Treatment of the reconstituted alpha(2A)-AR membranes containing Gbeta1gamma2his:Galpha(i1) with imidazole confirmed the specificity for the Ni2+:G-protein surface dissociation of Galpha(i1) from Gbeta1gamma2his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Conformational changes in the amino-terminal helix of the G protein alpha(i1) following dissociation from Gbetagamma subunit and activation

G protein alpha subunits mediate activation of signaling pathways through G protein-coupled receptors (GPCR) by virtue of GTP-dependent conformational rearrangements. It is known that regions of disorder in crystal structures can be indicative of conformational flexibility within a molecule, and there are several such regions in G protein alpha subunits. The amino-terminal 29 residues of Galpha are alpha-helical only in the heterotrimer, where they contact the side of Gbeta, but little is known about the conformation of this region in the active GTP bound state. To address the role of the Galpha amino-terminus in G-protein activation and to investigate whether this region undergoes activation-dependent conformational changes, a site-directed cysteine mutagenesis study was carried out. Engineered Galpha(i1) proteins were created by first removing six native reactive cysteines to yield a mutant Galpha(i1)-C3S-C66A-C214S-C305S-C325A-C351I that no longer reacts with cysteine-directed labels. Several cysteine substitutions along the amino-terminal region were then introduced. All mutant proteins were shown to be folded properly and functional. An environmentally sensitive probe, Lucifer yellow, linked to these sites showed a fluorescence change upon interaction with Gbetagamma and with activation by AlF(4)(-). Other fluorescent probes of varying charge, size, and hydrophobicity linked to amino-terminal residues also revealed changes upon activation with bulkier probes reporting larger changes. Site-directed spin-labeling studies showed that the N-terminus of the Galpha subunit is dynamically disordered in the GDP bound state, but adopts a structure consistent with an alpha-helix upon interaction with Gbetagamma. Interaction of the resulting spin-labeled Galphabetagamma with photoactivated rhodopsin, followed by rhodopsin-catalyzed GTPgammaS binding, caused the amino-terminal domain of Galpha to revert to a dynamically disordered state similar to that of the GDP-bound form. Together these results suggest conformational changes occur in the amino-termini of Galpha(i) proteins upon subunit dissociation and upon activating conformational changes. These solution studies reveal insights into conformational changes that occur dynamically in solution.     

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.     

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.     

Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i)

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.     

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.