The GoLoco motif: heralding a new tango between G protein signaling and cell division

The Galpha and Gbetagamma components of heterotrimeric G proteins, typically associated with cell-surface receptor signaling, also partake in the macromolecular interactions that underlie cell polarity and cell division. Proteins with Galpha-binding GoLoco motifs, such as Drosophila melanogaster Pins (for Partner of Inscuteable) and its mammalian counterpart LGN, participate in multi-protein complexes that maintain cellular asymmetry and orderly segregation of chromosomal content and daughter cell bodies. The GoLoco motif was recently identified as a selective Galpha-binding partner: the GoLoco-Galpha interaction can displace Gbetagamma and inhibit guanine nucleotide release from the bound Galpha subunit. Recent x-ray crystallographic studies suggest ways in which GoLoco-motif peptides may modulate heterotrimeric G protein signaling. Such peptides could be exploited to help dissect the signals that underpin cell polarity and cell division processes.     

Assembly and trafficking of heterotrimeric G proteins

To be activated by cell surface G protein-coupled receptors, heterotrimeric G proteins must localize at the cytoplasmic surface of plasma membranes. Moreover, some G protein subunits are able to traffic reversibly from the plasma membrane to intracellular locations upon activation. This current topic will highlight new insights into how nascent G protein subunits are assembled and how they arrive at plasma membranes. In addition, recent reports have increased our knowledge of activation-induced trafficking of G proteins. Understanding G protein assembly and trafficking will lead to a greater understanding of novel ways that cells regulate G protein signaling.     

Asymmetrically distributed C. elegans homologs of AGS3/PINS control spindle position in the early embryo

BACKGROUND: Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric Galpha subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown. RESULTS: Here we show that the Galpha subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). Finally, we show that the GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3. CONCLUSIONS: Our results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins.     

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.     

The melanosomal/lysosomal protein OA1 has properties of a G protein‐coupled receptor

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell‐specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein‐coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR‐mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well‐established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.     

A Golgi-localized hexose transporter is involved in heterotrimeric G protein-mediated early development in Arabidopsis

Signal transduction involving heterotrimeric G proteins is universal among fungi, animals, and plants. In plants and fungi, the best understood function for the G protein complex is its modulation of cell proliferation and one of several important signals that are known to modulate the rate at which these cells proliferate is D-glucose. Arabidopsis thaliana seedlings lacking the beta subunit (AGB1) of the G protein complex have altered cell division in the hypocotyl and are D-glucose hypersensitive. With the aim to discover new elements in G protein signaling, we screened for gain-of-function suppressors of altered cell proliferation during early development in the agb1-2 mutant background. One agb1-2-dependent suppressor, designated sgb1-1(D) for suppressor of G protein beta1 (agb1-2), restored to wild type the altered cell division in the hypocotyl and sugar hypersensitivity of the agb1-2 mutant. Consistent with AGB1 localization, SGB1 is found at the highest steady-state level in tissues with active cell division, and this level increases in hypocotyls when grown on D-glucose and sucrose. SGB1 is shown here to be a Golgi-localized hexose transporter and acts genetically with AGB1 in early seedling development.     

Non-canonical signaling and localizations of heterotrimeric G proteins

Heterotrimeric G proteins typically transduce signals from G protein-coupled receptors (GPCRs) to effector proteins. In the conventional G protein signaling paradigm, the G protein is located at the cytoplasmic surface of the plasma membrane, where, after activation by an agonist-bound GPCR, the GTP-bound Gα and free Gβγ bind to and regulate a number of well-studied effectors, including adenylyl cyclase, phospholipase Cβ, RhoGEFs and ion channels. However, research over the past decade or more has established that G proteins serve non-canonical roles in the cell, whereby they regulate novel effectors, undergo activation independently of a GPCR, and/or function at subcellular locations other than the plasma membrane. This review will highlight some of these non-canonical aspects of G protein signaling, focusing on direct interactions of G protein subunits with cytoskeletal and cell adhesion proteins, the role of G proteins in cell division, and G protein signaling at diverse organelles.     

Implications of non-canonical G-protein signaling for the immune system

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which consist of three subunits α, β, and γ, function as molecular switches to control downstream effector molecules activated by G protein-coupled receptors (GPCRs). The GTP/GDP binding status of Gα transmits information about the ligand binding state of the GPCR to intended signal transduction pathways. In immune cells heterotrimeric G proteins impact signal transduction pathways that directly, or indirectly, regulate cell migration, activation, survival, proliferation, and differentiation. The cells of the innate and adaptive immune system abundantly express chemoattractant receptors and lesser amounts of many other types of GPCRs. But heterotrimeric G-proteins not only function in classical GPCR signaling, but also in non-canonical signaling. In these pathways the guanine exchange factor (GEF) exerted by a GPCR in the canonical pathway is replaced or supplemented by another protein such as Ric-8A. In addition, other proteins such as AGS3-6 can compete with Gβγ for binding to GDP bound Gα. This competition can promote Gβγ signaling by freeing Gβγ from rapidly rebinding GDP bound Gα. The proteins that participate in these non-canonical signaling pathways will be briefly described and their role, or potential one, in cells of the immune system will be highlighted.     

Arabidopsis G‐protein interactome reveals connections to cell wall carbohydrates and morphogenesis

The heterotrimeric G‐protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G‐protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G‐protein associates with heptahelical G‐protein‐coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G‐protein effectors and scaffold proteins, we screened a set of proteins from the G‐protein complex using two‐hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G‐protein interactome. Within this core, over half of the interactions comprising two‐thirds of the nodes were retested and validated as genuine in planta. Co‐expression analysis in combination with phenotyping of loss‐of‐function mutations in a set of core interactome genes revealed a novel role for G‐proteins in regulating cell wall modification.     

Tubulin,actin and heterotrimeric G proteins: Coordination of signaling and structure

G proteins mediate signals from membrane G protein coupled receptors to the cell interior, evoking significant regulation of cell physiology. The cytoskeleton contributes to cell morphology, motility, division, and transport functions. This review will discuss the interplay between heterotrimeric G protein signaling and elements of the cytoskeleton. Also described and discussed will be the interplay between tubulin and G proteins that results in atypical modulation of signaling pathways and cytoskeletal dynamics. This will be extended to describe how tubulin and G proteins act in concert to influence various aspects of cellular behavior. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters.This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Localization of Gi alpha proteins in the centrosomes and at the midbody: implication for their role in cell division

At the plasma membrane, heterotrimeric G proteins act as molecular switches to relay signals from G protein-coupled receptors; however, G(alpha) subunits also have receptor-independent functions at intracellular sites. Regulator of G protein signaling (RGS) 14, which enhances the intrinsic GTPase activity of G(ialpha) proteins, localizes in centrosomes, which suggests the coexpression of G(ialpha). We show expression of G(ialpha1), G(ialpha2), and G(ialpha3) in the centrosomes and at the midbody. Fluorescence resonance energy transfer analysis confirms a direct interaction between RGS14 and G(ialpha1) in centrosomes. Expression of GTPase-deficient G(ialpha1) results in defective cytokinesis, whereas that of wild-type or GTPase-deficient G(ialpha3) causes prolonged mitosis. Cells treated with pertussis toxin, with reduced expression of G(ialpha1), G(ialpha2), and G(ialpha3) or with decreased expression of RGS14 also exhibit cytokinesis defects. These results suggest that G(ialpha) proteins and their regulators at these sites may play essential roles during mammalian cell division.     

Receptor- and nucleotide exchange-independent mechanisms for promoting G protein subunit dissociation

Mechanisms for heterotrimeric G protein activation that do not rely on G protein coupled receptor activation are becoming increasingly apparent. We recently identified beta gamma subunit-binding peptides that we proposed bound to a "hot spot" on beta gamma subunits, stimulating G protein dissociation without stimulating nucleotide exchange and activating G protein signaling in intact cells. AGS3, a member of the activators of G protein signaling family of proteins, also activates G protein signaling in a nucleotide exchange-independent manner, and AGS3 homologues are involved in asymmetric cell division during development. Here we demonstrate that a consensus G protein regulatory (GPR) peptide from AGS3 and related proteins is sufficient to induce G protein subunit dissociation and that both the GPR and hot spot-binding peptides promote dissociation to extents comparable with a known G protein activator, AMF. Peptides derived from adenylyl cyclase 2 and GRK2 prevented formation of the heterotrimeric complex but did not alter the rate of alpha subunit dissociation from beta gamma subunits. These data indicate that these nucleotide exchange-independent G protein activator peptides do not simply compete for alpha interactions with beta gamma subunits, but actively promote subunit dissociation. Thus, we propose two novel mechanisms for nucleotide exchange independent activation of G protein signaling, one that involves conformational changes in the alpha subunit and one that involves conformational changes in the beta gamma subunits.     

Drosophila neuroblast asymmetric cell division: recent advances and implications for stem cell biology

Asymmetric cell division is an evolutionarily conserved mechanism widely used to generate cellular diversity during development. Drosophila neuroblasts have been a useful model system for studying the molecular mechanisms of asymmetric cell division. In this minireview, we focus on recent progress in understanding the role of heterotrimeric G proteins and their regulators in asymmetric spindle geometry, as well as the role of an Inscuteable-independent microtubule pathway in asymmetric localization of proteins in neuroblasts. We also discuss issues of progenitor proliferation and differentiation associated with asymmetric cell division and their broader implications for stem cell biology.     

The melanosomal/lysosomal protein OA1 has properties of a G protein-coupled receptor

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein-coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR-mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well-established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.     

The beta-subunit of G proteins is a substrate of protein histidine phosphatase

Increasing evidence suggests that reversible phosphorylation of histidine residues in proteins is important for signaling cascades in eukaryotic cells. Recently, the first eukaryotic protein histidine phosphatase (PHP) was identified. The beta1-subunit of heterotrimeric G proteins (Gbeta) undergoes phosphorylation on His266 which is apparently involved in receptor-independent G protein activation. We studied whether phosphorylated Gbeta-subunits are substrates of PHP. Phosphorylated Gbetagamma dimers of the retinal G protein transducin and Gbeta in membrane preparations of H10 cells (neonatal rat cardiomyocytes) were dephosphorylated by PHP. Overexpression of PHP in H10 cells showed that PHP and Gbeta also interfere within cells. In membranes of cells overexpressing PHP, the amount of phosphorylated Gbeta was largely reduced. Both our in vitro and cell studies indicate that phosphorylated Gbeta-subunits of heterotrimeric G proteins are substrates of PHP. Therefore, PHP might play a role in the regulation of signal transduction via heterotrimeric G proteins.     

Heterotrimeric G protein signaling in cancer cells with regard to metastasis formation

The signal transduction mediated by heterotrimeric G proteins is involved in the regulation of a plethora of cell functions ranging from the sensation of light, taste and odor to chemotaxis, inflammation and the coordination of immune responses. These reactions have in common that they occur fast and are short-lived. Apart from this, it becomes increasingly evident, that the signaling of heterotrimeric G proteins has an imminent function in gene regulation, too, and therefore mediates even long-term effects. Herein, we illustrate the pathways of the four classes of α subunits and of the βγ subunits of these heterotrimeric G proteins especially with regard to their function in cancer. G protein signaling is crucial for the development and localization of metastases and furthermore has been shown to be involved in tumor growth and angiogenesis. We summarize the current knowledge, how these processes are regulated by the short-term cellular response and the long-term gene regulation in cancer cells, and we discuss possible strategies for a therapeutic intervention.     

Heterotrimeric G proteins and regulation of size asymmetry during cell division

The generation of daughter cells of different fate and size depends on the orientation, positioning and morphology of the mitotic spindle. In both C. elegans and Drosophila, heterotrimeric G proteins have emerged as central and conserved regulators of this process. Although the same molecular players are involved in worms and flies, there are clear differences in the mechanisms used. Interestingly, recent work in mammalian cells suggests that heterotrimeric G proteins may control spindle positioning in higher organisms during symmetric and asymmetric cell divisions.     

Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division

Drosophila melanogaster neuroblasts (NBs) undergo asymmetric divisions during which cell-fate determinants localize asymmetrically, mitotic spindles orient along the apical-basal axis, and unequal-sized daughter cells appear. We identified here the first Drosophila mutant in the Ggamma1 subunit of heterotrimeric G protein, which produces Ggamma1 lacking its membrane anchor site and exhibits phenotypes identical to those of Gbeta13F, including abnormal spindle asymmetry and spindle orientation in NB divisions. This mutant fails to bind Gbeta13F to the membrane, indicating an essential role of cortical Ggamma1-Gbeta13F signaling in asymmetric divisions. In Ggamma1 and Gbeta13F mutant NBs, Pins-Galphai, which normally localize in the apical cortex, no longer distribute asymmetrically. However, the other apical components, Bazooka-atypical PKC-Par6-Inscuteable, still remain polarized and responsible for asymmetric Miranda localization, suggesting their dominant role in localizing cell-fate determinants. Further analysis of Gbetagamma and other mutants indicates a predominant role of Partner of Inscuteable-Galphai in spindle orientation. We thus suggest that the two apical signaling pathways have overlapping but different roles in asymmetric NB division.     

GTP-binding proteins in plants: new members of an old family

Regulatory guanine nucleotide-binding proteins (G proteins) have been studied extensively in animal and microbial organisms, and they are divided into the heterotrimeric and the small (monomeric) classes. Heterotrimeric G proteins are known to mediate signal responses in a variety of pathways in animals and simple eukaryotes, whiole small G proteins perform diverse functions including signal transduction, secretion, and regulation of cytoskeleton. In recent years, biochemical analyses have produced a large amount of information on the presence and possible functions of G proteins in plants. Further, molecular cloning has clearly demonstrated that plants have both heterotrimeric and small G proteins. Although the functions of the plant heterotrimeric G proteins are yet to be determined, expression analysis of an Arabidopsis G protein suggests that it may be involved in the regulation of cell division and differentiation. In contrast to the very few genes cloned thus far that encode heterotrimeric G proteins in plants, a large number of small G proteins have been identified by molecular cloning from various plants. In addition, several plant small G proteins have been shown to be functional homologues of their counterparts in animals and yeasts. Future studies using a number of approaches are likely to yield insights into the role plant G proteins play.