The G-protein-coupled receptor GCR1 regulates DNA synthesis through activation of phosphatidylinositol-specific phospholipase C

Different lines of evidence suggest that specific events during the cell cycle may be mediated by a heterotrimeric G-protein activated by a cognate G-protein coupled receptor. However, coupling between the only known Galpha-subunit of the heterotrimeric G-protein (GPA1) and the only putative G-protein coupled receptor (GCR1) of plants has never been shown. Using a variety of approaches, we show here that GCR1-enhanced thymidine incorporation into DNA depends on an increase in phosphatidylinositol-specific phospholipase C activity and an elevation of inositol 1,4,5-trisphosphate levels in the cells. Tobacco (Nicotiana tabacum) cells that overexpress either Arabidopsis GCR1 or GPA1 display this phenomenon. We suggest on the basis of these results that GCR1-controlled events during the cell cycle involve phosphatidylinositol-specific phospholipase C as an effector of GCR1 and inositol 1,4,5-trisphosphate as a second messenger, and that GCR1 and GPA1 are both involved in this particular signaling pathway.     

In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: possible involvement of lipid raft

Regulators of G-protein signaling (RGS) are a family of proteins which accelerate intrinsic GTP-hydrolysis on heterotrimeric G-protein-alpha-subunits. Although it has been suggested that the function of RGS4 is reciprocally regulated by competitive binding of the membrane phospholipid, phosphatidylinositol-3,4,5,-trisphosphate(PtdIns(3,4,5)P(3)), and Ca(2+)/calmodulin (CaM), it remains to be shown that these interactions occur in vivo. Here, using fluorescence resonance energy transfer (FRET) techniques, we show that an elevation of intracellular Ca(2+) concentration by ionomycin increased the FRET efficiency from ECFP (a variant of cyan fluorescent protein)-labeled calmodulin to Venus (a variant of yellow fluorescent protein)-labeled RGS4. The increase in FRET efficiency was greatly attenuated by pre-treating the cells with methyl-beta-cyclodextrin, which depletes membrane cholesterol and thus disrupts lipid rafts. These results provide the first demonstration of a Ca(2+)-dependent interaction between RGS4 and CaM in vivo and show that association in lipid rafts of the plasma membrane might be involved in this physiological regulation of RGS proteins.     

cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin synthesis in Arabidopsis

While studying blue light-independent effects of cryptochrome 1 (cry1) photoreceptor, we observed premature opening of the hook in cry1 mutants grown in complete darkness, a phenotype that resembles the one described for the heterotrimeric G-protein α subunit (GPA1) null mutant gpa1. Both cry1 and gpa1 also showed reduced accumulation of anthocyanin under blue light. These convergent gpa1 and cry1 phenotypes required the presence of sucrose in the growth media and were not additive in the cry1 gpa1 double mutant, suggesting context-dependent signaling convergence between cry1 and GPA1 signaling pathways. Both, gpa1 and cry1 mutants showed reduced GTP-binding activity. The cry1 mutant showed wild-type levels of GPA1 mRNA or GPA1 protein. However, an anti-transducin antibody (AS/7) typically used for plant Gα proteins, recognized a 54?kDa band in the wild type but not in gpa1 and cry1 mutants. We propose a model where cry1-mediated post-translational modification of GPA1 alters its GTP-binding activity.     

The mechanism of membrane-translocation of regulator of G-protein signaling (RGS) 8 induced by Galpha expression

RGS (regulators of G-protein signaling) proteins comprise a large family that modulates heterotrimeric G-protein signaling. This protein family has a common RGS domain and functions as GTPase-activating proteins for the alpha-subunits of heterotrimeric G-proteins located at the plasma membrane. RGS8 was identified as a neuron-specific RGS protein, which belongs to the B/R4 subfamily. We previously showed that RGS8 protein was translocated to the plasma membrane from the nucleus on coexpression of GTPase-deficient Galphao (GalphaoQL). Here, we first examined which subtypes of Galpha can induce the translocation of RGS8. When the Galphai family was expressed, the translocation of RGS8 did occur. To investigate the mechanism of this translocation, we generated a mutant RGS8 with reduced affinity to Galphao and an RGS-insensitive (RGS-i) mutant of GalphaoQL. Co-expression experiments with both mutants revealed that disruption of the Galpha-RGS8 interaction abolished the membrane-translocation of RGS8 despite the apparent membrane localization of RGS-i GalphaoQL. These results demonstrated that RGS8 is recruited to the plasma membrane where G-proteins are activated mainly by direct association with Galpha.     

G-protein complex mutants are hypersensitive to abscisic acid regulation of germination and postgermination development

Abscisic acid (ABA) plays regulatory roles in a host of physiological processes throughout plant growth and development. Seed germination, early seedling development, stomatal guard cell functions, and acclimation to adverse environmental conditions are key processes regulated by ABA. Recent evidence suggests that signaling processes in both seeds and guard cells involve heterotrimeric G proteins. To assess new roles for the Arabidopsis (Arabidopsis thaliana) Galpha subunit (GPA1), the Gbeta subunit (AGB1), and the candidate G-protein-coupled receptor (GCR1) in ABA signaling during germination and early seedling development, we utilized knockout mutants lacking one or more of these components. Our data show that GPA1, AGB1, and GCR1 each negatively regulates ABA signaling in seed germination and early seedling development. Plants lacking AGB1 have greater ABA hypersensitivity than plants lacking GPA1, suggesting that AGB1 is the predominant regulator of ABA signaling and that GPA1 affects the efficacy of AGB1 execution. GCR1 acts upstream of GPA1 and AGB1 for ABA signaling pathways during germination and early seedling development: gcr1 gpa1 double mutants exhibit a gpa1 phenotype and agb1 gcr1 and agb1 gcr1 gpa1 mutants exhibit an agb1 phenotype. Contrary to the scenario in guard cells, where GCR1 and GPA1 have opposite effects on ABA signaling during stomatal opening, GCR1 acts in concert with GPA1 and AGB1 in ABA signaling during germination and early seedling development. Thus, cell- and tissue-specific functional interaction in response to a given signal such as ABA may determine the distinct pathways regulated by the individual members of the G-protein complex.     

Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.     

Arabidopsis nucleoside diphosphate kinase-2 as a plant GTPase activating protein

Nucleoside diphosphate kinase (NDPK) is involved in multiple signaling pathways in mammalian systems, including G-protein signaling. Arabidopsis NDPK2, like its mammalian counterparts, is multifunctional despite its initial discovery phytochrome-interacting protein. This similarity raises the possibility that NDPK2 may play a role in G-protein signaling in plants. In the present study, we explore the potential relationship between NDPK2 and the small G proteins, Pra2 and Pra3, as well as the heterotrimeric G protein, GPA1. We report a physical interaction between NDPK2 and these small G proteins, and demonstrate that NDPK2 can stimulate their GTPase activities. Our results suggest that NDPK2 acts as a GTPase-activating protein for small G proteins in plants. We propose that NDPK2 might be a missing link between the phytochromemediated light signaling and G protein-mediated signaling.     

RGS1 is expressed in monocytes and acts as a GTPase-activating protein for G-protein-coupled chemoattractant receptors.

The leukocyte response to chemoattractants is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). RGS1 is a member of a protein family constituting a newly appreciated and large group of proteins that act as deactivators of G-protein signaling pathways by accelerating the GTPase activity of G-protein alpha subunits. We demonstrate here that RGS1 is expressed in human monocytes; by immunofluorescence and subcellular fractionation RGS1 was localized to the plasma membrane. By using a mixture of RGS1 and plasma membranes, we were able to demonstrate GAP activity of RGS1 on receptor-activated G-proteins; RGS1 did not affect ligand-stimulated GDP-GTP exchange. We found that RGS1 desensitizes a variety of chemotactic receptors including receptors for N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and C5a. Interaction of RGS proteins and ligand-induced G-protein signaling can be demonstrated by determining GTPase activity using purified RGS proteins and plasma membranes.     

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.     

Ligand-induced partitioning of human CXCR1 chemokine receptors with lipid raft microenvironments facilitates G-protein-dependent signaling

Ligand binding to a chemokine receptor triggers signaling events through heterotrimeric G-proteins. The mechanisms underlying receptor-mediated G-protein activation in the heterogeneous microenvironments of the plasma membrane are unclear. Here, using live-cell fluorescence resonance energy transfer imaging to detect the proximity between CXCR1-cyan fluorescent protein (CFP) and fluorescence probes that label lipid raft or non-lipid raft microdomains and using fluorescence recovery after photobleaching analysis to measure the lateral diffusion of CXCR1-CFP, we found that interleukin-8 induces association between the receptors and lipid raft microenvironments. Disruption of lipid rafts impaired G-protein-dependent signaling, such as Ca2+ responses and phosphatidylinositol 3-kinase activation, but had no effect on ligand-binding function and did not completely abolish ligand-induced receptor phosphorylation. Our results suggest a novel mechanism by which ligand binding to CXCR1 promotes lipid raft partitioning of receptors and facilitates activation of heterotrimeric G-proteins.     

Heterotrimeric G protein alpha and beta subunits antagonistically modulate stomatal density in Arabidopsis thaliana

Stomata are essential for efficient gas and water-vapor exchange between the atmosphere and plants. Stomatal density and movement are controlled by a series of signal molecules including phytohormones and peptides as well as by environmental stimuli. It is known that heterotrimeric G-proteins play an important role in the ABA-inhibited stomatal opening. In this study, the G-protein signaling pathway was also found to regulate stomatal density on the lower epidermis of Arabidopsis cotyledons. The loss-of-function mutation of the G-protein α-subunit (GPA1) showed a reduction in stomatal density, while overexpression of the constitutively active form of GPA1^QL increased stomatal density, indicating a positive role of the active form of GPA1 in stomatal development. In contrast, stomatal density increased in the null mutant of the G-protein β-subunit (AGB1) but decreased in transgenic lines that overexpressed AGB1. Stomatal analysis of the gpa1 agb1 double mutants displayed an average value of stomatal density compared to the single mutants. Taken together, these results suggest that the stomatal density in Arabidopsis is modulated by GPA1 and AGB1 in an antagonistic manner.     

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration

Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Galpha(i)/Galpha(o) heterotrimeric signaling pathways. In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Galpha(i)/Galpha(o). AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.     

Use of G-protein fusions to monitor integral membrane protein-protein interactions in yeast

The control of protein-protein interactions is a fundamental aspect of cell regulation. Here we describe a new approach to detect the interaction of two proteins in vivo. By this method, one binding partner is an integral membrane protein whereas the other is soluble but fused to a G-protein gamma-subunit. If the binding partners interact, G-protein signaling is disrupted. We demonstrate interaction between known binding partners, syntaxin 1a with neuronal Sec1 (nSec1), and the fibroblast-derived growth factor receptor 3 (FGFR3) with SNT-1. In addition, we describe a genetic screen to identify nSec1 mutants that are expressed normally, but are no longer able to bind to syntaxin 1a. This provides a convenient method to study interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods.     

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.     

Heterotrimeric G-protein signaling in Arabidopsis: Puzzling G-protein-coupled receptor

Seven transmembrane G-protein-coupled receptors (GPCRs) are commonly used by eukaryotes to sense extracellular signals to switch on cellular responses through the activation of cognate heterotrimeric G-proteins. In Arabidopsis thaliana, GCR2 has been proposed as a GPCR for the plant hormone abscisic acid. On the other hand, biochemical analysis demonstrates that the sole Arabidopsis heterotrimeric G-protein α subunit, GPA1, is in the activated state (GTP-bound) by default, suggesting that the heterotrimeric G-proteins may act without any GPCRs.Key words: heterotrimeric G-proteins, GCR2, GPA1, G-protein-coupled receptor (GPCR), AtRGS1     

Differential Role of Axin RGS Domain Function in Wnt Signaling during Anteroposterior Patterning and Maternal Axis Formation

Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.     

Differential roles of Arabidopsis heterotrimeric G-protein subunits in modulating cell division in roots

Signaling through heterotrimeric G proteins is conserved in diverse eukaryotes. Compared to vertebrates, the simpler repertoire of G-protein complex and accessory components in Arabidopsis (Arabidopsis thaliana) offers a unique advantage over all other multicellular, genetic-model systems for dissecting the mechanism of G-protein signal transduction. One of several biological processes that the G-protein complex regulates in Arabidopsis is cell division. We determined cell production rate in the primary root and the formation of lateral roots in Arabidopsis to define individually the types of modulatory roles of the respective G-protein alpha- and beta-subunits, as well as the heterotrimer in cell division. The growth rate of the root is in part a consequence of cell cycle maintenance in the root apical meristem (RAM), while lateral root production requires meristem formation by founder pericycle cells. Thus, a comparison of these two parameters in various genetic backgrounds enabled dissection of the role of the G-protein subunits in modulation of cell division, both in maintenance and initiation. Cell production rates were determined for the RAM and lateral root formation in gpa1 (Arabidopsis G-protein alpha-subunit) and agb1 (Arabidopsis G-protein beta-subunit) single and double mutants, and in transgenic lines overexpressing GPA1 or AGB1 in agb1 or gpa1 mutant backgrounds, respectively. We found in the RAM that the heterotrimeric complex acts as an attenuator of cell proliferation, whereas the GTP-bound form of the Galpha-subunit's role is a positive modulator. In contrast, for the formation of lateral roots, the Gbetagamma-dimer acts largely independently of the Galpha-subunit to attenuate cell division. These results suggest that Arabidopsis heterotrimeric G-protein subunits have differential and opposing roles in the modulation of cell division in roots.     

Heterotrimeric G�� subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositide-specific phospholipase C, PI-PLC1

The canonical Gα subunit of the heterotrimeric G protein complex from wheat (Triticum aestivum), GA3, and the calcium-binding protein, Clo3, were revealed to interact both in vivo and in vitro and Clo3 was shown to enhance the GTPase activity of GA3. Clo3 is a member of the caleosin gene family in wheat with a single EF-hand domain and is induced during cold acclimation. Bimolecular Fluorescent Complementation (BiFC) was used to localize the interaction between Clo3 and GA3 to the plasma membrane (PM). Even though heterotrimeric G-protein signaling and Ca2? signaling have both been shown to play a role in the response to environmental stresses in plants, little is known about the interaction between calcium-binding proteins and Gα. The GAP activity of Clo3 towards GA3 suggests it may play a role in the inactivation of GA3 as part of the stress response in plants. GA3 was also shown to interact with the phosphoinositide-specific phospholipase C, PI-PLC1, not only in the PM but also in the endoplasmic reticulum (ER). Surprisingly, Clo3 was also shown to interact with PI-PLC1 in the PM and ER. In vitro analysis of the protein-protein interaction showed that the interaction of Clo3 with GA3 and PI-PLC1 is enhanced by high Ca2? levels. Three-way affinity characterizations with GA3, Clo3 and PI-PLC1 showed the interaction with Clo3 to be competitive, which suggests that Clo3 may play a role in the Ca2?-triggered feedback regulation of both GA3 and PI-PLC1. This hypothesis was further supported by the demonstration that Clo3 has GAP activity with GA3.     

The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo

Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.     

Re-evaluation of the cytokinin receptor role of the Arabidopsis gene GCR1

GCR1 has been tentatively identified inArabidopsis thaliana as the first plant G-protein coupled receptor (GPCR) (Josefsson and Rask 1997) implicated in the cytokinin sensory pathway (Plakidou-Dymock et al. 1998). A protein fusion of GCR1 and green fluorescent protein has been expressed inArabidopsis and shown GCR1 to be located on the plasma membrane. Studies of plants with altered GCR1 expression have led us to question GCR1's involvement in cytokinin signaling. TransgenicArabidopsis plants containing sense and antisense constructs for GCR1 have been produced and over- and under-expression confirmed. The analysis of 12 antisense and 17 sense lines has failed to reveal the previously reported »Dainty« phenotype or altered cytokinin sensitivity. We have used the »Gauntlet« approach to test the plants' response to various plant hormones although this has not yet identified a mutant phenotype. The yeast-two hybrid system has been used and so far there is no evidence to suggest GCR1 interacts with heterotrimeric G proteins. Before GCR1 can be identified as genuine G-protein coupled receptor, the identification of a ligand and a proof of association with heterotrimeric G-proteins should be obtained.