Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gα(q) and Gα(s) regulation of neurotransmission, Gα(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and Gα(olf)-dependent odorant receptor signaling. Mammalian Ric-8 activities are partitioned between two genes, ric-8A and ric-8B. Ric-8A is a guanine nucleotide exchange factor (GEF) for Gα(i)/α(q)/α(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a Gα(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be Gα subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed Gα(s) and lesser amounts of Gα(q) and Gα(13). Ric-8BFL stimulated guanosine 5'-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gα(olf), whereas the Ric-8BΔ9 isoform stimulated Gα(s short) GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of Gα(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to Gα(s) from ～385 nm to an estimated value of ～42 μM. Directionality of the Ric-8BFL-catalyzed Gα(s) exchange reaction was GTP-dependent. At sub-K(m) GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gα(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·Gα nucleotide-free intermediate to release free Ric-8B and Gα-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce Gα-GTP in cells.     

Ric-8 regulation of heterotrimeric G proteins

Abstract

Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein α subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the Gα subunits that each binds in vitro and seemingly regulate diverse G protein signaling systems in cells. Combined evidence from worm, fly and mammalian systems has shown that Ric-8 proteins are required to maintain proper cellular abundances of G proteins. Ric-8 proteins support G protein levels by serving as molecular chaperones that promote Gα subunit biosynthesis. In this review, the evidence that Ric-8 proteins act as non-receptor GEF activators of G proteins in signal transduction contexts will be weighed against the evidence supporting the molecular chaperoning function of Ric-8 in promoting G protein abundance. I will conclude by suggesting that Ric-8 proteins may act in either capacity in specific contexts. The field awaits additional experimentation to delineate the putative multi-functionality of Ric-8 towards G proteins in cells.     

The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα?GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1?GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1?GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1?GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1?GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.     

Levels of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclase isoforms (1, 5/6, 8) in post-mortem human brain caudate and cortical membranes: comparison with rat brain membranes and potential stoichiometric relationships

The levels of expression of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclases isoforms (AC1, 5/6, and 8) in human brain cortical and caudate membranes were quantified by western blot analysis in order to establish their contribution to the patterns of AC functioning. Both areas expressed Gsα(long) (52 kDa) with values ranging from about 1400 ng/mg of membrane protein in cerebral cortex to close to 600 ng/mg of membrane protein in caudate nucleus. In contrast, Gsα(short) and Gsα(olf) were expressed separately, Gsα(short) in cortical membranes with values around 500 ng/mg of membrane protein and Gα(olf) in caudate membranes with values around 1300 ng/mg of membrane protein. Quantitative measurements of Gβ, revealed a similar expression level in cortical and caudate membranes (5444±732 versus 5511±394 ng/mg protein; p=0.966). The B(max) values of GTPγS-dependent [(3)H]-forskolin binding show the following descending order: rat striatal membranes>rat cortical membranes=human caudate membranes>human cortical membranes. Therefore, as measured immunochemically and by [(3)H]-forskolin binding, there seems to be a vast excess of Gsα subunits over catalytic units of AC. The highest levels of AC5/6 expression were detected in caudate membranes. AC8 was little expressed, and there were no significant differences in the relative values between both human brain regions. Finally, the levels of the AC1 isoform were significantly lower in caudate than in cortical membranes. It is concluded that these stoichiometric data contribute nonetheless to explain the significant differences observed in signalling capacities through the AC system in both human brain regions.     

Ric-8A,a GEF,and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.     

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.     

Backbone resonance assignments for G protein αi3 subunit in the GTP-bound state

Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein α subunit (Gα(GDP)) and G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα(GTP)) and Gβγ. Then, Gα(GTP) and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Gα_i3, a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPγS.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Activation of adenylyl cyclases, regulation of insulin status, and cell survival by G(alpha)olf in pancreatic beta-cells

Because we recently identified the G(alpha)olf subunit in rat pancreatic beta-cells, we investigated the downstream effectors and the biological functions of this G protein in HEK-293T cells and the insulin-secreting mouse betaTC-3 cell line. With the use of transient transfection of HEK-293T cells with constitutively activated G(alpha)olf (G(alpha)olfQ214L, i.e., AG(alpha)olf), together with expression vectors encoding the adenylyl cyclase (AC) isoforms (AC-I to -VIII and soluble AC), compared with cotransfections using AG(alphas) (G(alphas)R201C), we observed that AG(alpha)olf preferentially activates AC-I and -VIII, which are also expressed in beta-cells. Stable overexpression of wild-type or AG(alpha)olf in betaTC-3 cells resulted in partial attenuation of insulin secretion and biosynthesis, suggesting that chronic activation of the G(alpha)olf-signaling pathway is associated with beta-cell desensitization. In agreement, transfected betaTC-3 cells present a decreased insulin content with respect to parental cells, whereas the proinsulin convertases PC-1 and PC-2 were unaffected. Furthermore, betaTC-3-AG(alpha)olf cells are resistant to serum starvation-induced apoptosis. Our findings suggest that G(alpha)olf is involved in insulin status, cell survival, and regeneration of the insulin-secreting beta-cells during development and diabetes.     

Backbone resonance assignments for G protein αi3 subunit in the GDP-bound state

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP–GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gα_i3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα_i3 in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα_i3·GDP would be useful for the analyses of the dynamics of Gα_i3 and its interactions with various target molecules.     

Ca2+-dependent GTPase, extra-large G protein 2 (XLG2), promotes activation of DNA-binding protein related to vernalization 1 (RTV1), leading to activation of floral integrator genes and early flowering in Arabidopsis

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, play important roles in plant development and cell signaling. In Arabidopsis, in addition to one prototypical G protein α subunit, GPA1, there are three extra-large G proteins, XLG1, XLG2, and XLG3, of largely unknown function. Each extra-large G (XLG) protein has a C-terminal Gα-like region and a ～400 amino acid N-terminal extension. Here we show that the three XLG proteins specifically bind and hydrolyze GTP, despite the fact that these plant-specific proteins lack key conserved amino acid residues important for GTP binding and hydrolysis of GTP in mammalian Gα proteins. Moreover, unlike other known Gα proteins, these activities require Ca(2+) instead of Mg(2+) as a cofactor. Yeast two-hybrid library screening and in vitro protein pull-down assays revealed that XLG2 interacts with the nuclear protein RTV1 (related to vernalization 1). Electrophoretic mobility shift assays show that RTV1 binds to DNA in vitro in a non-sequence-specific manner and that GTP-bound XLG2 promotes the DNA binding activity of RTV1. Overexpression of RTV1 results in early flowering. Combined overexpression of XLG2 and RTV1 enhances this early flowering phenotype and elevates expression of the floral pathway integrator genes, FT and SOC1, but does not repress expression of the floral repressor, FLC. Chromatin immunoprecipitation assays show that XLG2 increases RTV1 binding to FT and SOC1 promoters. Thus, a Ca(2+)-dependent G protein, XLG2, promotes RTV1 DNA binding activity for a subset of floral integrator genes and contributes to floral transition.     

Switch-domain mutations in the Saccharomycescerevisiae G protein α-subunit Gpa1p identify a receptor subtype-biased mating defect

The response to pheromone in Saccharomyces cerevisiae involves a heterotrimeric G protein composed of Gpa1p (α subunit), Ste4p (β) and Ste18p (γ). The switch II region of Gα subunits is involved in several protein-protein interactions and an intrinsic GTPase activity. To investigate the role of this region of Gpa1p, we have analyzed the effect of switch II mutations. The Q323 analog in Gα subunits and Ras is implicated in GTP hydrolysis. Mutation of the Q323 residue of Gpa1p resulted in constitutive activation of the pheromone response pathway and eliminated the ability to interact with Ste4p, consistent with a defect in GTPase activity. Mutation of residue A59 of Ras and the analogous Gαs residue has had quite different effects. The analogous Gpa1p G321T mutation resulted in phenotypes consistent with a less severe GTPase defect, but also led to an unexpected mating phenotype: mating was decreased in both mating types, but the defect was 1000-fold more severe in α cells than in a cells. In addition the G321T mutation resulted in an unusual pheromone response phenotype. We discuss the possibility that these phenotypes may reflect a differential role for the switch II region in activation by the a- and α-factor receptors.     

Genetic and structural analysis of G protein alpha subunit regulatory domains.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.     

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.     

Resistance to inhibitors of cholinesterase-8A (Ric-8A) is critical for growth factor receptor-induced actin cytoskeletal reorganization

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that Gα(13), a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These Gα(13)-mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to Gα(13) is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to Gα(13). Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified Gα(13) protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of Gα(13) to the cell cortex. Hence, Ric-8A is critical for growth factor receptor-induced actin cytoskeletal reorganization.     

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.     

Ric-8A catalyzes guanine nucleotide exchange on G alphai1 bound to the GPR/GoLoco exchange inhibitor AGS3

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Galpha subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Galpha.GDP subunits and forms a stable nucleotide-free Ric-8A:Galpha complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Galpha subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Galpha.GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Galpha(il) and that of Ric-8A with the AGS3-C:Galpha(il).GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Galpha(i1).GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Galpha(i1).GDP. Subsequent dissociation of AGS3-C and GDP from Galpha(i1) yields a stable nucleotide free Ric-8A.Galpha(i1) complex that, in the presence of GTP, dissociates to yield Ric-8A and Galpha(i1).GTP. AGS3-C does not induce dissociation of the Ric-8A.Galpha(i1) complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Galpha(i1).GDP ensures unidirectional activation of Galpha subunits that cannot be reversed by AGS3.     

A dominant-negative Galpha mutant that traps a stable rhodopsin-Galpha-GTP-betagamma complex

Residues comprising the guanine nucleotide-binding sites of the α subunits of heterotrimeric (large) G-proteins (Gα subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both Gα subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric Gα subunit (designated αT*) that is mainly comprised of the α subunit of the retinal G-protein transducin and a limited region from the α subunit of Gi1. The αT*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type αT*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on αT*(S43N). The αT*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type αT*. Thus, GTP-bound αT*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. αT*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type αT*. Activation of the PDE by αT*(S43N) was unaffected if either R* or β1γ1 alone was present, whereas it was inhibited when R* and the β1γ1 subunit were added together. Overall, our studies suggest that the S43N substitution on αT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound Gα subunit, and the β1γ1 complex.     

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.