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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner

Arap3 is a phosphoinositide (PI) 3 kinase effector that serves as a GTPase activating protein (GAP) for both Arf and Rho G-proteins. The protein has multiple pleckstrin homology (PH) domains that bind preferentially phosphatidyl-inositol-3,4,5-trisphosphate (PI(3,4,5,)P3) to induce translocation of Arap3 to the plasma membrane upon PI3K activation. Arap3 also contains a Ras association (RA) domain that interacts with the small G-protein Rap1 and a sterile alpha motif (SAM) domain of unknown function. In a yeast two-hybrid screen for new interaction partners of Arap3, we identified the PI 5'-phosphatase SHIP2 as an interaction partner of Arap3. The interaction between Arap3 and SHIP2 was observed with endogenous proteins and shown to be mediated by the SAM domain of Arap3 and SHIP2. In vitro, these two domains show specificity for a heterodimeric interaction. Since it was shown previously that Arap3 has a higher affinity for PI(3,4,5,)P3 than for PI(3,4)P2, we propose that the SAM domain of Arap3 can function to recruit a negative regulator of PI3K signaling into the effector complex.     

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.     

Non-receptor activators of heterotrimeric G-protein signaling (AGS proteins)

G-protein coupled receptor (GPCR) signaling represents one of the most conserved and ubiquitous means in mammalian cells for transferring information across the plasma membrane to the intracellular environment. Heterotrimeric G-protein subunits play key roles in transducing these signals, and intracellular regulators influencing the activation state and interaction of these subunits regulate the extent and duration of GPCR signaling. One class of intracellular regulator, the non-receptor activators of G-protein signaling (or AGS proteins), are the major focus of this review. AGS proteins provide a basis for understanding the function of heterotrimeric G-proteins in both GPCR-driven and GPCR independent cellular signaling pathways.     

AGS proteins, GPR motifs and the signals processed by heterotrimeric G proteins

A long term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation by heterotrimeric G-proteins (G). G-proteins play a central role in cellular communication mediating the cell response to numerous hormones and neurotransmitters. A major determinant of signalling specificity for heterotrimeric G-proteins is the cell specific expression of the subtypes of the primary signalling entities, receptor, G and effector (E). Another major site for regulating signalling specificity lies at the R-G or G-E interface where these interactions are influenced by cell architecture, the stoichiometry of signalling components and accessory proteins that may segregate the receptor to microdomains of the cell, regulate the efficiency and/or specificity of signal transfer and/or influence the activation state of G-protein independent of a classical G-protein coupled receptor. One strategy to address these issues in our laboratory involves the identification of cellular proteins that regulate the transfer of signal from receptor to G or directly influence the activation state of G independent of a classical G-protein coupled receptor. We identified three proteins, AGS1, AGS2 and AGS3 (for Activators of G-protein Signaling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1, 2 and 3 interact with different subunits and/or conformations of heterotrimeric G-proteins, selectively activate different G-proteins, provide unexpected mechanisms for regulation of the G-protein activation cycle and have opened up a new area of research related to the cellular role of G-proteins as signal transducers.     

Translocation of Activator of G-protein Signaling 3 to the Golgi Apparatus in Response to Receptor Activation and Its Effect on the trans-Golgi Network

Group II activators of G-protein signaling play diverse functional roles through their interaction with Gα_i, Gα_t, and Gα_o via a G-protein regulatory (GPR) motif that serves as a docking site for Gα-GDP. We recently reported the regulation of the AGS3-Gα_i signaling module by a cell surface, seven-transmembrane receptor. Upon receptor activation, AGS3 reversibly dissociates from the cell cortex, suggesting that it may function as a signal transducer with downstream signaling implications, and this question is addressed in the current report. In HEK-293 and COS-7 cells expressing the α_2A/D-AR and Gα_i3, receptor activation resulted in the translocation of endogenous AGS3 and AGS3-GFP from the cell cortex to a juxtanuclear region, where it co-localized with markers of the Golgi apparatus (GA). The agonist-induced translocation of AGS3 was reversed by the α₂-AR antagonist rauwolscine. The TPR domain of AGS3 was required for agonist-induced translocation of AGS3 from the cell cortex to the GA, and the translocation was blocked by pertussis toxin pretreatment or by the phospholipase Cβ inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Agonist-induced translocation of AGS3 to the GA altered the functional organization and protein sorting at the trans-Golgi network. The regulated movement of AGS3 between the cell cortex and the GA offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the trans-Golgi network.     

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.     

Physiological actions of regulators of G-protein signaling (RGS) proteins

Regulators of G-protein signaling (RGS) proteins are a family of proteins, which accelerate GTPase-activity intrinsic to the alpha subunits of heterotrimeric G-proteins and play crucial roles in the physiological control of G-protein signaling. If RGS proteins were active unrestrictedly, they would completely suppress various G-protein-mediated cell signaling as has been shown in the over-expression experiments of various RGS proteins. Thus, physiologically the modes of RGS-action should be under some regulation. The regulation can be achieved through the control of either the protein function and/or the subcellular localization. Examples for the former are as follows: (i) Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) inhibits RGS-action, which can be recovered by Ca(2+)/calmodulin. This underlies a voltage-dependent "relaxation" behavior of G-protein-gated K(+) channels. (ii) A modulatory protein, 14-3-3, binds to the RGS proteins phosphorylated by PKA and inhibits their actions. For the latter mechanism, additional regulatory modules, such as PDZ, PX, and G-protein gamma subunit-like (GGL) domains, identified in several RGS proteins may be responsible: (i) PDZ domain of RGS12 interacts with a G-protein-coupled chemokine receptor, CXCR2, and thus facilitates its GAP action on CXCR2-mediated G-protein signals. (ii) RGS9 forms a complex with a type of G-protein beta-subunit (Gbeta5) via its GGL domain, which facilitates the GAP function of RGS9. Both types of regulations synergistically control the mode of action of RGS proteins in the physiological conditions, which contributes to fine tunings of G-protein signalings.