Interaction of Activator of G-protein Signaling 3 (AGS3) with LKB1, a Serine/Threonine Kinase Involved in Cell Polarity and Cell Cycle Progression

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AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDPbound conformation of G␣ i > G␣ 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 (G␣ s -mediated vasoactive intestinal peptide receptor) and inhibitory (G␣ i -mediated ␣ 2 -adrenergic receptor (␣ 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 ␣ 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 G␣ i -coupled receptor. Incubation of cells with the ␣ 2 -AR agonist UK14304 (1 M) for 18 h resulted in a ϳ1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated G␣ i3 . Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of G␣ 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 G␣ i in the membrane, which influences the adaptation of the cell to prolonged activation of G␣ i -coupled receptors.The activation/deactivation cycle for heterotrimeric G-protein involves the exchange of GDP for GTP and the subsequent hydrolysis of GTP by G␣ subunits. These events are generally initiated by a G-protein-coupled receptor at the cell surface. However, increasing evidence indicates the existence of unexpected regulatory mechanisms for the G-protein activation/ deactivation cycle and a role for G-protein subunits in signaling events within the cell that do not involve a cell-surface receptor (1). These unexpected regulatory mechanisms include accessory proteins that accelerate GTPase activity, serve as novel binding partners for G␣ and G␤␥ subunits, and/or regulate guanine nucleotide exchange independent of a G-protein coupled receptor (1-6).

Section: Resultsmentioning

confidence: 68%

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

Sato

Gettys

Lanier

2004

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“…According to the mammalian heterotrimeric G-protein paradigm, G␣ in the GDP-bound resting state associates with G␤␥, whereas G␣ in the GTP-bound activated state dissociates from G␤␥ (8). Some activators of Gprotein signaling proteins identified in other organisms have been shown to bind to the resting or GDP-bound form of G␣ to release G␤␥ (28,29). The present results demonstrate that PLD␣1-G␣ interaction occurs with or without added GDP, and thus, PLD␣1 binds to the resting state, GDP-bound G␣.…”

Section: Discussionmentioning

confidence: 99%

Arabidopsis Phospholipase Dα1 Interacts with the Heterotrimeric G-protein α-Subunit through a Motif Analogous to the DRY Motif in G-protein-coupled Receptors

Zhao

Wang

2004

171

135

Phospholipase D (PLD) and heterotrimeric G-protein both play important, diverse roles in cellular regulation and signal transduction. Here we have determined the physical interaction between plant PLD and the only canonical ␣-subunit (G␣) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction. PLD␣1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with G␣. PLD␣1 contains a sequence motif analogous to the G␣-interacting DRY motif normally conserved in G-protein-coupled receptors. Mutation of the central Lys residue PLD K564A of this motif abolished the PLD␣1-G␣ binding, whereas mutation of the two flanking residues PLD E563A and PLD F565A decreased the binding. Addition of G␣ to PLD␣1 inhibited PLD␣1 activity, whereas the PLD K564A mutation that disrupted the G␣-PLD␣1 binding abolished the inhibition. GTP relieved the G␣ inhibition of PLD␣1 activity and also inhibited the binding between PLD␣1 and G␣. Meanwhile, the PLD␣1-G␣ interaction stimulated the intrinsic GTPase activity of G␣. Therefore, these results have demonstrated the direct binding between G␣ and PLD␣1, identified the DRY motif on PLD␣1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLD␣1 and G␣.Phospholipase D (PLD), 1 which hydrolyzes phospholipids to phosphatidic acid and a head group, plays diverse roles in cellular metabolism and regulation. Plant PLD comprises a family of enzymes with different regulatory properties (1). Several Arabidopsis PLDs have been shown to display different requirements for Ca 2ϩ , polyphosphoinositides, and free fatty acids as well as varied substrate selectivity. Arabidopsis has at least 12 PLDs, of which PLD␣1 is most prevalent and responsible for the common plant PLD activity (1, 2). PLD␣1 produces a majority of the phosphatic acid under several stress conditions, such as freezing and wounding (3, 4). Suppression of PLD␣1 delayed abscisic acid (ABA)-promoted senescence (5), decreased wound-induced accumulation of jasmonic acid (4) and reactive oxygen generation (6), and increased freezing tolerance and water loss (3, 7). These results show that the common plant PLD has multifaceted functions, including roles in metabolism and cell signaling, dependent on the nature and severity of the stress conditions.The G␣ subunit of heterotrimeric G-proteins plays an important role in signal transduction. In animal systems, G␣ interacts with the upstream transmembrane G-protein-coupled receptors (GPCRs) and with the ␤-subunit. The binding of a ligand to a cognate receptor promotes the exchange of GDP for GTP on G␣, and the GTP-bound G␣ activates the downstream effector proteins (8). In addition, G␣ may interact with nonreceptor proteins to mediate signaling (9). Mammalian cells contain a number of G␣s that mediate many distinctive cellular functions (8). In contrast, the number of G␣s is very limited in plants; Arabidopsis has only a single canonical G␣ gene, GPA1 (10, 11). Arabidopsis G␣-null mutants are impaired i...

“…The fragment of AGS4/G18.1b in the latter immunoprecipitates was actually phosphorylated at Ser-59, which is just upstream of the first GPR motif and analogous to the site of phosphorylation in RGS14 upstream of the GPR motif, where it was suggested to influence the affinity of the interaction of the GPR motif with G-protein (24). Additional sites of phosphorylation may occur within the GPR motif itself, influencing the interaction with G-protein and providing a regulatory mechanism for signal input or subcellular location (25).…”

Section: Figmentioning

confidence: 99%

“…In this regard, AGS4/G18.1b, AGS3, and LGN are similar in that each of the proteins contain multiple GPR motifs downstream of an apparent regulatory domain (tetratricopeptide repeats for AGS3 and LGN) (10) that regulates the subcellular location of the protein and/or possibly influences the interaction of the GPR motifs with G-proteins via interaction with binding partners (5,19,25,(27)(28)(29)(30). The presence of multiple GPR motifs allow the protein to bind more than one G␣ subunit (25) along with whatever other proteins may complex at the amino-terminal region of the proteins (25,27). Such complexing of proteins may be key for turning on or off the signal or play a role in the spatial dynamics of signaling events.…”

Section: Figmentioning

confidence: 99%

Identification and Characterization of AGS4

Cao

Cismowski

Sato

et al. 2004

Self Cite

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.