Thermodynamic Characterization of the Binding of Activator of G Protein Signaling 3 (AGS3) and Peptides Derived from AGS3 with Gαi1

Adhikari, Anirban; Sprang, Stephen R.

doi:10.1074/jbc.m306300200

Cited by 52 publications

(57 citation statements)

References 36 publications

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“…Three core GPR motifs are present at the carboxyl-terminal region of AGS4/G18.1b (GPR-I Thr-62-Arg-81, GPR-II Arg-104 -Arg-123, GPR-III Gln-133-Arg-152). Four GPR motifs are found in AGS3 and LGN, where at least for AGS3, it appears that each of the four GPR motifs can simultaneously bind G i ␣GDP free of G␤␥ (10,26). Structurefunction studies with a consensus GPR peptide (15) and the crystal structure of the RGS14-GPR/GoLoco peptide complexed with G i ␣ 1 (16) provide important structural information on key residues required for interaction with and regulation of G i ␣.…”

Section: Figmentioning

confidence: 99%

Identification and Characterization of AGS4

Cao

Cismowski

Sato

et al. 2004

Journal of Biological Chemistry

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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 guaninenucleotide exchange by G i ␣. 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 ␣, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5 -3-O-(thio)triphosphate (GTP␥S) binding to purified G i ␣ 1 . AGS4/G18.1b was also complexed with G i ␣ 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 G␤␥-regulated effector phospholipase C-␤2. 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 ␣ independent of G␤␥ participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.

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Section: Figmentioning

confidence: 99%

Identification and Characterization of AGS4

Cao

Cismowski

Sato

et al. 2004

Journal of Biological Chemistry

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“…For this work, we have used a synthetic short form of AGS3 (AGS3-C) that encodes all four GPR/GoLoco motifs (35). Earlier thermodynamic studies have shown that all four GPR/GoLoco motifs of AGS3-C bind cooperatively to G␣ i1 ⅐GDP and form a stable AGS3-C:[G␣ i1 ⅐GDP] 4 complex (40). For simplicity, we henceforth refer to this complex as AGS3-C:G␣ i1 ⅐GDP.…”

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confidence: 99%

Ric-8A Catalyzes Guanine Nucleotide Exchange on Gαi1 Bound to the GPR/GoLoco Exchange Inhibitor AGS3

Thomas

Tall

Adhikari

et al. 2008

Journal of Biological Chemistry

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Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including G␣ 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 G␣⅐GDP subunits and forms a stable nucleotide-free Ric-8A:G␣ complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes G␣ subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for G␣⅐GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:G␣ il and that of Ric-8A with the AGS3-C:G␣ il ⅐GDP 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:G␣ i1 ⅐GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:G␣ i1 ⅐GDP. Subsequent dissociation of AGS3-C and GDP from G␣ i1 yields a stable nucleotide free Ric-8A⅐G␣ i1 complex that, in the presence of GTP, dissociates to yield Ric-8A and G␣ i1 ⅐GTP. AGS3-C does not induce dissociation of the Ric-8A⅐G␣ i1 complex, even when present at very high concentrations. The action of Ric-8A on AGS3:G␣ i1 ⅐GDP ensures unidirectional activation of G␣ subunits that cannot be reversed by AGS3.Canonical G-protein signaling pathways are activated when agonist-bound heptahelical receptors, acting as guanine nucleotide exchange factors (GEFs), 2 promote the exchange of GDP for GTP on G␣ subunits present in G␣⅐GDP:G␤␥ heterotrimers (1-3). Upon binding GTP, conformational changes in the switch regions of G␣ subunits destabilize the heterotrimer and allow G␣⅐GTP to dissociate from G␤␥ subunits (4, 5). Downstream regulatory molecules such as the regulators of G-protein signaling (RGS) accelerate G␣-catalyzed GTP hydrolysis, allowing the G␣ subunits to revert to their resting GDP-bound conformation and priming them for the next receptor-induced G-protein cycle (6 -8). Receptor-mediated signaling accounts for the majority of G-protein-regulated cellular control mechanisms. However, during the past few years evidence has emerged that, in both lower and higher eukaryotes, multicomponent G-protein signaling systems, operating outside the realm of membrane-bound receptors, play significant roles in various biological processes (9). These include control of the generation of microtubule pulling forces during cell division (10 -16), synaptic signaling processes (17), and cardiovascular function (18). A receptor-independent G-protein-mediated signaling pathway, regulating a fundamental event such as asymmetric cell division, may involve proteins that can modulate G-protein nucleotide exchange in a manner that resembles the action of agonist-bound receptors and G␤␥ subunits. In nematodes, asymmetric cell division is a result of eccentr...

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“…Individual GPR motifs may differ in their selectivity for individual Gαi isoforms Mittal and Linder, 2004;Shu et al, 2006) and regions outside of the core GPR motif can influence G-protein isoform selectivity (Adhikari and Sprang, 2003;Kimple et al, 2002;Kimple et al, 2004). AGS3-6 and other GPR-containing proteins provide an unexpected mode of regulation for signals involving Gi and Go G-proteins that is distinct from that achieved with the super family of GPCRs Sato et al, 2006a).…”

Section: Group II Ags Proteinsmentioning

confidence: 99%

“…The interaction of GPR motifs with Gαi/o stabilizes the GDP-bound conformation of Gα and interferes with Gβγ for binding to Giα . The ability of a protein with multiple GPR motifs to bind an equivalent number of multiple Gαi/o at any given moment presents an unusual scaffolding potential for organizing a signaling cassette (Adhikari and Sprang, 2003;Bernard et al, 2001;Tall and Gilman, 2005). It will be of great interest to define the structural aspects of a multiple GPR motif protein in which each motif is docked with a Gα subunit.…”

Section: Group II Ags Proteinsmentioning

confidence: 99%

“…The GPR motif consists of a core 20-25 amino acid sequence in which specific residues and spatial relationships within this sequence play an important role in the interaction with Giα ( Figure 5) (Peterson et al, 2002). Additional sequences outside of this core motif may influence selectivity for different Gα subunits and the relative affinities of individual GPR motifs for Gα isoforms (Adhikari and Sprang, 2003;Cao et al, 2004;Kimple et al, 2002;Kimple et al, 2004;McCudden et al, 2005;Mittal and Linder, 2004;Willard et al, 2006a;Willard et al, 2006b). The following sections will discuss current thoughts regarding GPR-mediated regulation of G-proteins, the regulation of selected GPR proteins and their role in cell division.…”

Section: Group II Ags Proteinsmentioning

confidence: 99%

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Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling

Blumer

Smrcka

Lanier

2007

Pharmacology & Therapeutics

122

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Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents, plays an important role in adaptive processes of organs, and aberrant processing of signals through these transducing systems is a component of various disease states. In addition to GPCR-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Gαβγ heterotrimer or Gα and Gαβγ subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Gα and Gαβγ) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Gαβγ. Such regulatory accessory proteins include the family of RGS proteins that accelerate the GTPase activity of Gα and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor independent activators of G-protein signaling or AGS proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways and provide a platform for diverse functions of both the heterotrimeric Gαβγ and the individual Gα and Gαβγ subunits.

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Thermodynamic Characterization of the Binding of Activator of G Protein Signaling 3 (AGS3) and Peptides Derived from AGS3 with Gαi1

Cited by 52 publications

References 36 publications

Identification and Characterization of AGS4

Identification and Characterization of AGS4

Ric-8A Catalyzes Guanine Nucleotide Exchange on Gαi1 Bound to the GPR/GoLoco Exchange Inhibitor AGS3

Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling

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