Nonreceptor Guanine Nucleotide Exchange Factor Research Articles

G Protein Binding Sites on Calnuc (Nucleobindin 1) and NUCB2 (Nucleobindin 2) Define a New Class of Gαi-regulatory Motifs

Heterotrimeric G proteins are molecular switches modulated by families of structurally and functionally related regulators. GIV (Gα-interacting vesicle-associated protein) is the first non-receptor guanine nucleotide exchange factor (GEF) that activates Gα(i) subunits via a defined, evolutionarily conserved motif. Here we found that Calnuc and NUCB2, two highly homologous calcium-binding proteins, share a common motif with GIV for Gα(i) binding and activation. Bioinformatics searches and structural analysis revealed that Calnuc and NUCB2 possess an evolutionarily conserved motif with sequence and structural similarity to the GEF sequence of GIV. Using in vitro pulldown and competition assays, we demonstrate that this motif binds preferentially to the inactive conformation of Gα(i1) and Gα(i3) over other Gα subunits and, like GIV, docks onto the α3/switch II cleft. Calnuc binding was impaired when Lys-248 in the α3 helix of Gα(i3) was replaced with M, the corresponding residue in Gα(o), which does not bind to Calnuc. Moreover, mutation of hydrophobic residues in the conserved motif predicted to dock on the α3/switch II cleft of Gα(i3) impaired the ability of Calnuc and NUCB2 to bind and activate Gα(i3) in vitro. We also provide evidence that calcium binding to Calnuc and NUCB2 abolishes their interaction with Gα(i3) in vitro and in cells, probably by inducing a conformational change that renders the Gα(i)-binding residues inaccessible. Taken together, our results identify a new type of Gα(i)-regulatory motif named the GBA motif (for Gα-binding and -activating motif), which is conserved across different proteins throughout evolution. These findings provide the structural basis for the properties of Calnuc and NUCB2 binding to Gα subunits and its regulation by calcium ions.

Ric-8a Catalyzed G Protein Activation Proceeds Through a Disordered State

Members of the Ras superfamily of regulatory GTP binding proteins, Heterotrimeric G protein alpha subunits (Ga) undergo cycles of activation and deactivation driven by binding and hydrolysis of GTP. Activation occurs by replacement of GDP by GTP at the nucleotide binding site of Ga, which requires catalytic assistance from guanine nucleotide exchange factors (GEFs). Transmembrane G protein-coupled Receptors (GPCRs) are the best known G protein GEFs, but recently, a novel family of cytosolic, non-receptor GEFs, typified by mammalian Ric-8A, were discovered. Unlike GPCRs, which act on G protein heterotrimers, Ric-8A catalyzes the release of GDP directly upon Gi-class Ga subunits (Gai), and has negligible affinity for Gai-GTP. Upon binding to Gai-GDP, Ric-8A catalyzes GDP release and forms a stable Gai:Ric-8A complex that dissociates only in the presence of GTP, resulting in the release of Gai-GTP. The TROSY-HSQC spectrum of [1H,15N]Gai bound to Ric-8A is considerably broadened relative to Gai-GDP. Hydrogen-deuterium exchange mass spectroscopy shows that the rate of HD exchange at Gai:Ric-8A is more than 2X faster than from Gai-GDP. Differential scanning calorimetry shows that both Ric-8A and Gai-GDP undergo cooperative, irreversible unfolding transitions at 47 deg and 52 deg, respectively, while nucleotide-free Gai shows a broad, weak transition near 35 deg. The unfolding transition for Gai:Ric-8A is complex, with a broad transition peaking at 49i. Ric-8A therefore stabilizes nucleotide-free Gai in a dynamic state, which, we propose, facilitates GTP binding. We show that the C-terminus of Gai is a critical binding element for Ric-8A, as is known to be the case for GPCRs, suggesting that these two GEFs act by similar mechanisms as chaperones for the unstable and dynamic nucleotide-free state of Ga.

Activation of the Regulator of G Protein Signaling 14−Gαi1-GDP Signaling Complex Is Regulated by Resistance to Inhibitors of Cholinesterase-8A

RGS14 is a brain scaffolding protein that integrates G protein and MAP kinase signaling pathways. Like other RGS proteins, RGS14 is a GTPase activating protein (GAP) that terminates Gαi/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds Gαi1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-Gαi1-GDP signaling complex to regulate its activation state. RGS14 and Ric-8A are recruited from the cytosol to the plasma membrane in the presence of coexpressed Gαi1 in cells, suggesting formation of a functional protein complex with Gαi1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex to form a stable Ric-8A-Gαi complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-Gαi1-GDP complex to stimulate both the steady-state GTPase activity of Gαi1 and binding of GTP to Gαi1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on Gαi1 nucleotide binding and GTPase activity. This observation correlates with findings that show RGS14 and Ric-8A share an overlapping binding region within the last 11 amino acids of Gαi1. As further evidence that these proteins are functionally linked, native RGS14 and Ric-8A coexist within the same hippocampal neurons. These findings demonstrate that RGS14 is a newly appreciated integrator of unconventional Ric-8A and Gαi1 signaling.

A GDI (AGS3) and a GEF (GIV) regulate autophagy by balancing G protein activity and growth factor signals

Autophagy is the major catabolic process responsible for the removal of aggregated proteins and damaged organelles. Autophagy is regulated by both G proteins and growth factors, but the underlying mechanism of how they are coordinated during initiation and reversal of autophagy is unknown. Using protein-protein interaction assays, G protein enzymology, and morphological analysis, we demonstrate here that Gα-interacting, vesicle-associated protein (GIV, a. k. a. Girdin), a nonreceptor guanine nucleotide exchange factor for Gα(i3), plays a key role in regulating autophagy and that dynamic interplay between Gα(i3), activator of G-protein signaling 3 (AGS3, its guanine nucleotide dissociation inhibitor), and GIV determines whether autophagy is promoted or inhibited. We found that AGS3 directly binds light chain 3 (LC3), recruits Gα(i3) to LC3-positive membranes upon starvation, and promotes autophagy by inhibiting the G protein. Upon growth factor stimulation, GIV disrupts the Gα(i3)-AGS3 complex, releases Gα(i3) from LC3-positive membranes, enhances anti-autophagic signaling pathways, and inhibits autophagy by activating the G protein. These results provide mechanistic insights into how reversible modulation of Gα(i3) activity by AGS3 and GIV maintains the delicate equilibrium between promotion and inhibition of autophagy.

Move or Divide?

A nonreceptor guanine nucleotide exchange factor for Gα i interacts with EGFR to tip cells from proliferating to migrating.

A Gαi–GIV Molecular Complex Binds Epidermal Growth Factor Receptor and Determines Whether Cells Migrate or Proliferate

Cells respond to growth factors by either migrating or proliferating, but not both at the same time, a phenomenon termed migration-proliferation dichotomy. The underlying mechanism of this phenomenon has remained unknown. We demonstrate here that Galpha(i) protein and GIV, its nonreceptor guanine nucleotide exchange factor (GEF), program EGF receptor (EGFR) signaling and orchestrate this dichotomy. GIV directly interacts with EGFR, and when its GEF function is intact, a Galpha(i)-GIV-EGFR signaling complex assembles, EGFR autophosphorylation is enhanced, and the receptor's association with the plasma membrane (PM) is prolonged. Accordingly, PM-based motogenic signals (PI3-kinase-Akt and PLCgamma1) are amplified, and cell migration is triggered. In cells expressing a GEF-deficient mutant, the Galphai-GIV-EGFR signaling complex is not assembled, EGFR autophosphorylation is reduced, the receptor's association with endosomes is prolonged, mitogenic signals (ERK 1/2, Src, and STAT5) are amplified, and cell proliferation is triggered. In rapidly growing, poorly motile breast and colon cancer cells and in noninvasive colorectal carcinomas in situ in which EGFR signaling favors mitosis over motility, a GEF-deficient splice variant of GIV was identified. In slow growing, highly motile cancer cells and late invasive carcinomas, GIV is highly expressed and has an intact GEF motif. Thus, inclusion or exclusion of GIV's GEF motif, which activates Galphai, modulates EGFR signaling, generates migration-proliferation dichotomy, and most likely influences cancer progression.

Characterization of GIV‐Gαi3, a Non‐Receptor GEF‐G protein complex involved in cell migration and metastasis

G‐protein coupled receptors (GPCRs) are the canonical guanine nucleotide exchange factors (GEFs) that activate trimeric G proteins. In contrast to GPCRs, little is known about the structure or function of non‐receptor GEFs. We previously reported that GIV/Girdin is a non‐receptor GEF for Gαi‐subunits that promotes cell migration and tumor cell metastasis (PNAS, 2009, 106: 3178–83). We discovered an evolutionarily conserved GEF motif in GIV that forms a unique interface with the Gα‐subunit. Mutation of a key residue (F1685) within this motif disrupted the Gαi‐GIV interaction and made GIV GEF‐deficient. We have now extensively characterized the molecular basis for the interaction between GIV and Gαi3 and its functional consequences. We found that GIV and Gαi3 can form a stable 1:1 complex in which the rate of nucleotide exchange, but not GTP hydrolysis, by the G protein is enhanced. Based on the preferential binding of GIV to Gαi versus Gαo, we have identified W258 in the Gαi‐subunit as a critical determinant for GIV binding. Mutation of W258 to the corresponding F in Gαo rendered Gαi3 GEF‐insensitive but did not perturb interaction with other Gα binding partners, i.e., Gβγ, GPCRs, GDIs or GAPs. When either the GEF‐deficient GIV mutant F1685A or GIV‐insensitive Gαi3 mutant W258F were expressed in HeLa cells they failed to undergo cell migration and to promote Akt signaling. These findings provide valuable information on the structural and biochemical basis for the biological functions of GIV as a Gαi activator which may be useful in the design of small molecules to disrupt the Gαi‐GIV interface for therapeutic purposes.Support: NIH DKI7780 and CA100768 (M.G.F). Susan G. Komen KG080079 (M.G‐M). NIH/T32 DK07202 and RSA from American Gastroenterology Association (P.G). McNair Student Research Scholarship (JE).

Girding for migratory cues: roles of the Akt substrate Girdin in cancer progression and angiogenesis

Cell migration is a fundamental aspect of a multitude of physiological and pathological processes, including embryonic development, inflammation, angiogenesis, and cancer progression. A variety of proteins are essential for cell migration, but context-specific signaling pathways and promigratory proteins must now be identified for our understanding of cancer biology to continue to advance. In this review, we focus on the emerging roles of Girdin (also designated KIAA1212, APE, GIV, and HkRP1), a novel component of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway that is a core-signaling transduction pathway in cancer progression. Girdin is expressed in some types of cancer cells and immature endothelial cells, and is therefore at the crossroads of multiple intracellular processes, including reorganization of the actin cytoskeleton, endocytosis, and modulation of Akt activity, which ultimately lead to cancer invasion and angiogenesis. It also acts as a nonreceptor guanine nucleotide exchange factor (GEF) for Galphai proteins. A significant observation is that Girdin, although vital for cancer progression and postnatal vascular remodelling, is dispensable for cell migratory events during embryonic development. These findings suggest that Girdin and its interacting proteins are potential pharmaceutical targets for cancer therapies and pathological anigiogenesis, including tumor angiogenesis.

GIV is a Non‐Receptor GEF for Gαi with a Unique Motif that Regulates Akt Signaling

Heterotrimeric G proteins are molecular switches that control signal transduction. Ligand‐occupied G‐protein coupled receptors serve are the canonical guanine nucleotide exchange factors (GEFs) that activate heterotrimeric G proteins. A few unrelated non‐receptor GEFs have also been described, but little or nothing is known about their structure, mechanism of action or cellular functions in mammals. We have discovered that GIV/Girdin serves as a non‐receptor GEF for Gαi through an evolutionarily conserved motif that forms a unique interface with the Gα‐subunit. Using insights from the 3‐dimensional structure of this interface, we demonstrate that this GEF motif enhances Akt signaling via the G ??‐PI3K pathway and that mutational disruption of the Gαi‐GIV interface inhibits Akt enhancement and cell migration in cancer cells. Recently we also found that GIV is a metastasis‐related protein in carcinomas by virtue of its ability to promote unrestricted Akt activation. Thus, the novel regulatory motif described here provides the structural and biochemical basis for the pro‐metastatic features of GIV, making the functional disruption of the Gαi‐GIV interface a promising target for therapy against cancer metastasis.Supported by NIH grants CA100768 and DKI7780 to M.G.F, by Basque Government Postdoctoral fellowship BFI06.300 and Susan G. Komen Postdoctoral fellowship KG080079 to MG‐M and by training grant NIH/T32 DK07202 and a Research Scholar Award from American Gastroenterology Association (AGA) to PG.

GIV is a nonreceptor GEF for Gαi with a unique motif that regulates Akt signaling

Heterotrimeric G proteins are molecular switches that control signal transduction. Ligand-occupied, G protein-coupled receptors serve as the canonical guanine nucleotide exchange factors (GEFs) that activate heterotrimeric G proteins. A few unrelated nonreceptor GEFs have also been described, but little or nothing is known about their structure, mechanism of action, or cellular functions in mammals. We have discovered that GIV/Girdin serves as a nonreceptor GEF for G alpha i through an evolutionarily conserved motif that shares sequence homology with the synthetic GEF peptide KB-752. Using the available structure of the KB-752 x G alpha i1 complex as a template, we modeled the G alpha i-GIV interface and identified the key residues that are required to form it. Mutation of these key residues disrupts the interaction and impairs Akt enhancement, actin remodeling, and cell migration in cancer cells. Mechanistically, we demonstrate that the GEF motif is capable of activating as well as sequestering the G alpha-subunit, thereby enhancing Akt signaling via the G betagamma-PI3K pathway. Recently, GIV has been implicated in cancer metastasis by virtue of its ability to enhance Akt activity and remodel the actin cytoskeleton during cancer invasion. Thus, the novel regulatory motif described here provides the structural and biochemical basis for the prometastatic features of GIV, making the functional disruption of this unique G alpha i-GIV interface a promising target for therapy against cancer metastasis.

Coactivation of G Protein Signaling by Cell-Surface Receptors and an Intracellular Exchange Factor

G protein-coupled receptors (GPCRs) mediate responses to a broad range of chemical and environmental signals. In yeast, a pheromone-binding GPCR triggers events leading to the fusion of haploid cells. In general, GPCRs function as guanine-nucleotide exchange factors (GEFs); upon agonist binding, the receptor induces a conformational change in the G protein alpha subunit, resulting in exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) and in signal initiation. Signaling is terminated when GTP is hydrolyzed to GDP [1]. This well-established paradigm has in recent years been revised to include new components that rates of GDP release, GTP binding [2-8], and GTP hydrolysis[9, 10]. Here we report the discovery of a nonreceptor GEF, Arr4. Like receptors, Arr4 binds directly to the G protein,accelerates guanine-nucleotide exchange, and stabilizes the nucleotide-free state of the a subunit. Moreover, Arr4 promotes G protein-dependent cellular responses, including mitogen-activated protein kinase (MAPK) phosphorylation,new-gene transcription, and mating. In contrast to knownGPCRs, however, Arr4 is not a transmembrane receptor,but rather a soluble intracellular protein. Our data suggest that intracellular proteins function in cooperation with mating pheromones to amplify G protein signaling, thereby leading to full pathway activation.

Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins

Asymmetric division of Drosophila neuroblasts (NBs) and the Caenorhabditis elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, RIC-8, which has recently been characterized in C. elegans and in mammals. We show here that the Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pl cells. Ric-8 is necessary for membrane targeting of Galphai, Pins and Gbeta13F, presumably by regulating multiple Galpha subunit(s). Ric-8 forms an in vivo complex with Galphai and interacts preferentially with GDP-Galphai, which is consistent with Ric-8 acting as a GEF for Galphai. Comparisons of the phenotypes of Galphai, Ric-8, Gbeta13Fsingle and Ric-8;Gbeta13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Gai activity. In addition, Gbetagamma acts to restrict Galphai (and GoLoco proteins) to the apical cortex, where Galphai (and Pins) can mediate asymmetric spindle geometry.

Mammalian Ric-8A (Synembryn) Is a Heterotrimeric Gα Protein Guanine Nucleotide Exchange Factor

The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with Galpha(o) and Galpha(s) as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of Galpha proteins including Galpha(q), Galpha(i1), and Galpha(o), but not Galpha(s). The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound Galpha proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the Galpha protein; this complex dissociates upon binding of GTP to Galpha.