Gregory G. Tall

Ric-8A and Giα Recruit LGN, NuMA, and Dynein to the Cell Cortex To Help Orient the Mitotic Spindle

ABSTRACT In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein α (Gα) 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 Gαi 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 Gαi, thus preventing its GEF activity for Gαi. Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased Gαi 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.

Ric-8A Catalyzes Guanine Nucleotide Exchange on Gαi1 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 Gα 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 Gα·GDP subunits and forms a stable nucleotide-free Ric-8A:Gα complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Gα subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Gα·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:Gαil and that of Ric-8A with the AGS3-C:Gαil·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:Gαi1·GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Gαi1·GDP. Subsequent dissociation of AGS3-C and GDP from Gαi1 yields a stable nucleotide free Ric-8A·Gαi1 complex that, in the presence of GTP, dissociates to yield Ric-8A and Gαi1·GTP. AGS3-C does not induce dissociation of the Ric-8A·Gαi1 complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Gαi1·GDP ensures unidirectional activation of Gα subunits that cannot be reversed by AGS3.

Purification of heterotrimeric G protein α subunits by GST-Ric-8 association: Primary characterization of purified Gαolf

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gαolf for the first time and unprecedented yield of Gαq and Gα13. Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF4− (GTP mimicry) and found to be >80% pure, bind guanosine 5′-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gαolf showed that it binds GTPγS at a rate marginally slower than Gαs short and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gαs short.

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

ric-8 (resistance to inhibitors of cholinesterase 8) genes have positive roles in variegated G protein signaling pathways, including Gαq and Gαs regulation of neurotransmission, Gαi-dependent mitotic spindle positioning during (asymmetric) cell division, and Gαolf-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 Gαi/αq/α12/13 subunits. Ric-8B potentiated Gs signaling presumably as a Gαs-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 Gαs and lesser amounts of Gαq and Gα13. Ric-8BFL stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding to these subunits and Gαolf, whereas the Ric-8BΔ9 isoform stimulated Gαs short GTPγS binding only. Michaelis-Menten experiments showed that Ric-8BFL elevated the Vmax of Gαs steady state GTP hydrolysis and the apparent Km values of GTP binding to Gαs from ∼385 nm to an estimated value of ∼42 μm. Directionality of the Ric-8BFL-catalyzed Gαs exchange reaction was GTP-dependent. At sub-Km GTP, Ric-BFL was inhibitory to exchange despite being a rapid GDP release accelerator. Ric-8BFL binds nucleotide-free Gαs tightly, and near-Km 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.

Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits

We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A–immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4−–bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs 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 Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A–supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gβγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits.

Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner

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 Gi and Gq α-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 β-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.

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Background: IP3R forms homo- and heterotetrameric channels. However, the impact of the specific composition of the heterotetrameric channel is undefined. Results: Concatenated IP3R dimers formed functional tetrameric channels. Heterotetrameric channels containing IP3R1 and IP3R2 had identical properties to IP3R2. Conclusion: Heterotetrameric IP3R do not behave as a blend of the constituent monomers. Significance: This study represents the first demonstration of the properties of heterotetrameric IP3R of unambiguously defined composition. 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 Ca2+ release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca2+, 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 Ca2+ 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 Ca2+ 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.

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.

Structural Determinants Underlying the Temperature-sensitive Nature of a Gα Mutant in Asymmetric Cell Division of Caenorhabditis elegans

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Gαo) and GPA-16 (Gαi) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Gαi1(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Gα. At the permissive temperature, the mutant Gα was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Gα bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Gα subunit function in ACD.

Structures of Ric-8A, a G protein chaperone and activator

Ric-8A is a 60 kDa protein expressed in the cytoplasm of multicellular eukaryotic cells. Unrelated to G protein-coupled receptors, Ric-8A has in vitro activity as a guanine nucleotide exchange factor. As such, Ric-8A catalyzes the release of GDP of GDP from the alpha subunits of heterotrimeric G proteins (Ga), forming a nucleotide-free Ga:Ric8A complex. In the presence of GTP or non-hydrolyzable GP analogs, the complex dissociates, releasing Ga•GTP and Ric-8A. Ric-8A controls the cellular abundance of Ga by inhibiting its ubiquitination, and acting as a chaperone. The mechanisms by which Ric-8A carries out these essential activities are unknown.