Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein ? subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free G?-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein ? subunits are misfolded and degraded rapidly. Ric-8 proteins also act as G?-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial G? unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein-coupled receptors (GPCRs). G protein ?? is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/G? protein-protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies.
Phospholipase C? (PLC?) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on G??? heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLC?3. Both expressed and endogenous M3R interacted with PLC? in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLC?3. Deletion of the PLC?3 C terminus or deletion of the PLC?3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLC?3 plasma membrane localization. Purified PLC?3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLC?3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated G?q-dependent but not G??-dependent PLC?3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLC?3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLC?3 signaling beyond its canonical role as a guanine nucleotide exchange factor for G?.
Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ?1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.
We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein ? (G?) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during G? subunit translation and protein folding. G? translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4(-)-bound G?i, G?q, G?13, and G?s produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. G?i, G?q, and G?13, but not G?s produced from Ric-8A-depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A-depleted RRL enhanced GDP-AlF4(-)-bound G? subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated G?q was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of G?q that gel filtered as a ?100 kDa Ric-8A:G?q heterodimer. Addition of GTP?S to Ric-8A-supplemented WGE G?q translation resulted in dissociation of the Ric-8A:G?q heterodimer and production of functional G?q-GTP?S monomer. Excess G?? supplementation of WGE did not support functional G?q production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein ? subunits.
Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein ? subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the G? 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 G? 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.
Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric guanine nucleotide-binding protein (G protein) signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A- or Ric-8B-deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A(-/-) ES cells, which resulted from reduced steady-state amounts of G?(i), G?(q), and G?(13) proteins to <5% of those of wild-type cells. The amounts of G?(s) and total G? protein were partially reduced in Ric-8A(-/-) cells compared to those in wild-type cells, and only the amount of G?(s) was reduced substantially in Ric-8B(-/-) cells. The abundances of mRNAs encoding the G protein ? subunits were largely unchanged by loss of Ric-8A or Ric-8B. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous G?(i) and G?(q) were efficiently translated in Ric-8A(-/-) cells but integrated into endomembranes poorly; however, the reduced amounts of G protein ? subunits that reached the membrane still bound to nascent G??. Finally, G?(i), G?(q), and G?(1) proteins exhibited accelerated rates of degradation in Ric-8A(-/-) cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent G? subunits with cellular membranes.
Heterotrimeric G protein ? subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of G?, 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 G? subunits. Ric-8A binds to G?•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free G?i1 is stable, but dissociates upon binding of GTP to G?i1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from G?i1, experiments were conducted to characterize the physical state of nucleotide-free G?i1 (hereafter referred to as G?i1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free G?i1 is more accessible to trypsinolysis than G?i1•GDP, but less so than G?i1[ ] alone. The TROSY-HSQC spectrum of [(15)N]G?i1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]G?i1•GDP. Hydrogen-deuterium exchange in G?i1[ ] bound to Ric-8A is 1.5-fold more extensive than in G?i1•GDP. Differential scanning calorimetry shows that both Ric-8A and G?i1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free G?i1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:G?i1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and G?i1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of G?i1 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 G?.
ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including G?(q) and G?(s) regulation of neurotransmission, G?(i)-dependent mitotic spindle positioning during (asymmetric) cell division, and G?(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 G?(i)/?(q)/?(12/13) subunits. Ric-8B potentiated G(s) signaling presumably as a G?(s)-class GEF activator, but no demonstration has shown Ric-8B GEF activity. Here, two Ric-8B isoforms were purified and found to be G? subunit GDP release factor/GEFs. In HeLa cells, full-length Ric-8B (Ric-8BFL) bound endogenously expressed G?(s) and lesser amounts of G?(q) and G?(13). Ric-8BFL stimulated guanosine 5-3-O-(thio)triphosphate (GTP?S) binding to these subunits and G?(olf), whereas the Ric-8B?9 isoform stimulated G?(s short) GTP?S binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the V(max) of G?(s) steady state GTP hydrolysis and the apparent K(m) values of GTP binding to G?(s) from ?385 nm to an estimated value of ?42 ?M. Directionality of the Ric-8BFL-catalyzed G?(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 G?(s) tightly, and near-K(m) GTP levels were required to dissociate the Ric-8B·G? nucleotide-free intermediate to release free Ric-8B and G?-GTP. Ric-8BFL-catalyzed nucleotide exchange probably proceeds in the forward direction to produce G?-GTP in cells.
G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) ?-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma ?-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes.
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 G?i/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds G?i1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-G?i1-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 G?i1 in cells, suggesting formation of a functional protein complex with G?i1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-G?i1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-G?i1-GDP complex to form a stable Ric-8A-G?i complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-G?i1-GDP complex to stimulate both the steady-state GTPase activity of G?i1 and binding of GTP to G?i1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on G?i1 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 G?i1. 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 G?i1 signaling.
Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein ? subunits. Co-expression of G? subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted G? protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein ? subunit purification that was applicable to all G? subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein G?(olf) for the first time and unprecedented yield of G?(q) and G?(13). G? subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·G? complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. G? subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5-[?-thio]triphosphate (GTP?S), and stimulate appropriate G protein effector enzymes. A primary characterization of G?(olf) showed that it binds GTP?S at a rate marginally slower than G?(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G?(s short).
In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein alpha (G alpha) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G alpha(i) function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G alpha(i), thus preventing its GEF activity for G alpha(i). Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G alpha(i) expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.
Group II activators of G-protein signaling (AGS) serve as binding partners for G?(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-G? signaling modules may be differentially regulated by cell surface receptors or by different nonreceptor guanine nucleotide exchange factors. We determined the effect of the nonreceptor guanine nucleotide exchange factors AGS1, GIV/Girdin, and Ric-8A on the interaction of two distinct GPR proteins, AGS3 and AGS4, with G?(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-G?(i1)-YFP and AGS4-Rluc-G?(i1)-YFP BRET were regulated by Ric-8A but not by G?-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and G?(i1)-YFP. The inhibitory regulation of GPR-G?(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-G?(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-G?(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-G?(i1)-YFP and AGS4-Rluc-G-G?(i1)-YFP BRET were observed in both pellet and supernatant subcellular fractions and were regulated by Ric-8A in both fractions. The regulation of the GPR-G?(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore G? expression in Ric8A(-/-) mouse embryonic stem cells, involved two helical domains at the carboxyl terminus of Ric-8A. These data indicate a dynamic interaction between GPR proteins, G?(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.
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