SummaryPhytohormone abscisic acid (ABA) regulates stress-responsive gene expression during vegetative growth, which is mediated largely by cis-elements sharing the ACGTGGC consensus. Although many transcription factors are known to bind the elements in vitro, only a few have been demonstrated to have in vivo functions and their specific roles in ABA/stress responses are mostly unknown. Here, we report that ABF2, an ABF subfamily member of bZIP proteins interacting with the ABA-responsive elements, is involved in ABA/stress responses. Its overexpression altered ABA sensitivity, dehydration tolerance, and the expression levels of ABA/stress-regulated genes. Furthermore, ABF2 overexpression promoted glucose-induced inhibition of seedling development, whereas its mutation impaired glucose response. The reduced sugar sensitivity was not observed with mutants of two other ABF family members, ABF3 and ABF4. Instead, these mutants displayed defects in ABA, salt, and dehydration responses, which were not observed with the abf2 mutant. Our data indicate distinct roles of ABF family members: whereas ABF3 and ABF4 play essential roles in ABA/stress responses, ABF2 is required for normal glucose response. We also show that ABF2 overexpression affects multiple stress tolerance.

The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D(2) receptors (D(2)Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for beta-arrestins was altered. Agonist-induced internalization of D(2)Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D(2)R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D(2)R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D(2)R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D(2)R-intracellular loop 2/3 provoked desensitization as in wild-type D(2)R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D(2)R. When dissociation between D(2)R and beta-arrestin was inhibited or when the expression of cellular beta-arrestins was decreased, agonist-induced desensitization of D(2)R did not occur, suggesting that dissociation from beta-arrestin is the main cellular process required for resensitization of D(2)R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with beta-arrestin plays a major role in agonist-induced desensitization.

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The effects of four tanshinones isolated from Tanshen (the root of Salvia miltiorrhiza Bunge, Labiatae) were tested for their inhibition of nitric oxide production in macrophage cells, and the underlying molecular mechanisms studied. Of the four tanshinones used, 15, 16-dihydrotanshinone-I, tanshinone-IIA and cryptotanshinone, but not tanshinone I, demonstrated significant inhibition of the LPS-induced nitric oxide production in RAW 264.7 cells, with calculated IC50 values of 5, 8, and 1.5 microM, respectively. Tanshinones exerted inhibitory activities on the LPS-induced nitric oxide production only when applied concurrently with LPS, and tanshinone-IIA and cryptotanshinone were found to inhibit LPS-induced NF-kappaB mobilization and extracellular-regulated kinase (ERK) activation, respectively. These results suggest that tanshinones inhibit LPS-induced nitric oxide generation by interfering with the initial stage of LPS-induced expression of certain genes. NF-kappaB and ERK could be the molecular targets for tanshinones for the inhibition of LPS-induced nitric oxide production in macrophage cells.

Together with G protein-coupled receptor (GPCR) kinases (GRKs) and ␤-arrestins, RGS proteins are the major family of molecules that control the signaling of GPCRs. The expression pattern of one of these RGS family members, RGS9-2, coincides with that of the dopamine D 3 receptor (D 3 R) in the brain, and in vivo studies have shown that RGS9-2 regulates the signaling of D2-like receptors. In this study, ␤-arrestin2 was found to be required for scaffolding of the intricate interactions among the dishevelled-EGL10-pleckstrin (DEP) domain of RGS9-2, G␤5, R7-binding protein (R7BP), and D 3 R. The DEP domain of RGS9-2, under the permission of ␤-arrestin2, inhibited the signaling of D 3 R in collaboration with G␤5. ␤-Arrestin2 competed with R7BP and G␤5 so that RGS9-2 is placed in the cytosolic region in an open conformation which is able to inhibit the signaling of GPCRs. The affinity of the receptor protein for ␤-arrestin2 was a critical factor that determined the selectivity of RGS9-2 for the receptor it regulates. These results show that ␤-arrestins function not only as mediators of receptor-G protein uncoupling and initiators of receptor endocytosis but also as scaffolding proteins that control and coordinate the inhibitory effects of RGS proteins on the signaling of certain GPCRs.The regulation of G protein-coupled receptors (GPCRs) involves various cellular events in different time frames, and the detailed regulatory mechanism can be unique for each receptor type and the signal it mediates. Much of our knowledge concerning the molecular basis of homologous desensitization of GPCRs is derived from studies of the ␤ 2 -adrenergic receptor (␤ 2 AR), in which GPCR kinases (GRKs) and ␤-arrestins play central roles. According to this working model, GRK-mediated receptor phosphorylation, followed by the association of ␤-arrestin, causes uncoupling of the GPCR from the G protein (16,18,30). However, the detailed molecular mechanism of this uncoupling of receptors from the G protein is unclear, aside from the simple idea that ␤-arrestins could physically interfere with the interaction between the receptor and G protein.Upon agonist binding, GPCRs stimulate the conversion of the inactive heterotrimeric GTP-binding protein GDP-G␣␤␥ to GTP-G␣ and G␤␥. The duration of the active state of the G protein, GTP-G␣, is regulated by two different cellular components, the weak GTPase activity of G␣ itself and the catalytic activity of GTPase-activating proteins (GAPs). Regulators of G protein signaling (RGS) act as GAPs for the heterotrimeric G protein ␣ subunit (49). More than 30 RGS proteins have been discovered over the last decade, and they are divided into 8 subfamilies (11,26,52).Among these RGS proteins, RGS2, RGS4, and RGS9-2 are known to be mutually related to the dopaminergic nervous system. It is known that the expression of the genes for RGS2 and RGS4 changes in response to dopaminergic stimulation (43, 44); however, the roles of RGS2 and RGS4 in the signaling and intracellular trafficking of D 2 R and D 3 R have not been...

ARIA is an ARM repeat protein that is a positive regulator of ABA response. To identify ARIA-interacting proteins, we conducted yeast two-hybrid screening. One of the positive clones obtained from the screen encoded a protein kinase, AtNEK6, which belongs to the NIMA (Never In Mitosis, gene A)-related kinase family. We analyzed AtNEK6 over-expression (OX) and knockout (KO) lines to investigate its in vivo function. The AtNEK6 OX lines grew slowly, whereas the KO line germinated and grew faster than wild type plants. AtNEK6 also affected ABA and stress responses. During seed germination, AtNEK6 OX lines were hypersensitive to ABA and high osmolarity, whereas its KO line was partially insensitive to ABA and high osmolarity. Previously, AtNEK6 was shown to be involved in epidermal cell morphogenesis. Our results indicate that AtNEK6 is also involved in plant growth regulation and responses to ABA and high osmolarity during the seed germination stage.

Classical G protein-coupled receptors (GPCRs) and canonical Wnt pathways were believed to use distinct signaling pathways. However, recent studies have shown that these two pathways interact each other by sharing several intermediate signaling components. Recent in vivo studies showed that antipsychotic drugs, which block dopamine D2-like receptors, increase the cellular levels of downstream signaling components of canonical Wnt pathways, such as dishevelled (Dvl), glycogen synthase kinase 3␤ (GSK3␤), and ␤-catenin. These results suggest that some functional interactions might exist between Wnt pathway and D2-like receptors. In this study, we show that among five different dopamine receptor subtypes, D 2 receptor (D 2 R) selectively inhibited the Wnt signaling, which was measured by lymphoid enhancing factor-1 (LEF-1)-dependent transcriptional activities. D 2 R-mediated inhibition of Wnt signaling was agonist-and G protein-independent and did not require receptor phosphorylation or endocytosis. D 2 R inhibited the LEF-1-dependent transcriptional activities, and this inhibitory activity was not affected by the inhibition of GSK-3␤, suggesting that D 2 R inhibited the Wnt signaling by acting on the downstream of GSK3␤. D 2 R directly interacted with ␤-catenin through the second and third loops, leading to a reduction of ␤-catenin distribution in the nucleus, resulting in an inhibition of LEF-1-dependent transcription. This is a novel mechanism for the regulation of canonical Wnt signaling by GPCRs, in which receptor proteins recruit ␤-catenin from cytosol to the plasma membrane, resulting in the decrement of the ␤-catenin/LEF-1-dependent transcription in the nucleus.

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