The effects of Ric-8 gene perturbation experiments on G protein functional abundances.
| Organism | Ric-8 Homolog | Ric-8 Perturbation | G protein(s) Assessed (Gene Name) | Experimental Findings | References |
|---|---|---|---|---|---|
| Mammalian Cultured Cell Lines (HEK293, HeLa, COS, MEF, NIH3T3, mouse embryonic stem cells) | Ric-8A | Overexpression | Gαi1 (GNAI1) | Transfected Gαi1-YFP levels were 5-fold higher in HEK293 cells cotransfected with Ric-8A. | Oner et al. (2013) |
| Overexpression | Gαi2 (GNAI2) | Agonist-stimulated Gαi2-dependent inhibition of cAMP levels was enhanced in HEK293 cells cotransfected with Ric-8A. | Fenech et al. (2009) | ||
| siRNA knockdown | Gαq (GNAQ) | HEK293 siRNA knockdown of Ric-8A reduced Gq-coupled GPCR-stimulated ERK activation. | Nishimura et al. (2006) | ||
| siRNA & shRNA knockdown | Gαi1 (GNAI1) | HeLa cell Ric-8A knockdown decreased Gαi1 levels and levels of Gαi1-dependent mitotic protein complexes at the plasma membrane. Metaphase spindle orientation was impaired. | Woodard et al. (2010) | ||
| shRNA knockdown | Gα13 (GNA13) | Ric-8A–knockdown in mouse embryonic fibroblasts reduced immunostained Gα13 signal at the plasma membrane. | Wang et al. (2011) | ||
| Overexpression | Gαi2 (GNAI2) Gαq (GNAQ) Gα12 (GNA12) | Ric-8A overexpression in COS cells increased Gαi2, Gαq, and Gα12 levels. Ric-8A overexpression mitigated overexpressed Gαi2 and Gαq ubiquitination and degradation. | Chishiki et al. (2013) | ||
| Ric-8B | shRNA knockdown, overexpression | Gαs (Gnas) | Ric-8B knockdown in NIH3T3 cells reduced Gαs levels. Ric-8B overexpression increased Gαs levels without affecting Gnas transcription. Ric-8B overexpression inhibited Gαs ubiquitination. | Nagai et al. (2010) | |
| Overexpression | Gαolf (GNAL) Gβ1 (GB1) Gγ13 (GNG13) | Ric-8B overexpression in HEK293 cells increased expressed Gαolf, Gβ1, and Gγ13 membrane abundance. | Kerr et al. (2008) | ||
| Overexpression | Gαolf (GNAL) | Odorant-stimulated cAMP production in odorant receptor– and Gαolf-transfected HEK293 cells was potentiated by Ric-8B co-overexpression. Ric-8B expression increased Gαolf levels. | Von Dannecker et al. (2006) | ||
| Overexpression | Gαolf (GNAL) | Ric-8B and GNAL cotransfection potentiated agonist-induced cAMP production from two Golf-coupled GPCRs. | Von Dannecker et al. (2005) | ||
| Overexpression | Gαs (GNAS) | Coexpression of Ric-8B with Gαs-coupled odorant receptors in HEK293 cells increased cAMP production. | Yoshikawa and Touhara (2009) | ||
| Ric-8A & Ric-8B | Overexpression | Gαi1 (GNAI1) Gαs (GNAS) | Ric-8A and Ric-8BFL increased Gαi-YFP and Gαs-YFP levels, respectively, in HEK293 cotransfection experiments. | Chan et al. (2011a) | |
| Transgenic gene deletions Ric-8A−/− or Ric-8B−/− | Gαi1/2 (Gnai1/2) Gαo (Gnao) Gαq (Gnaq) Gα13 (Gna13) Gβ1–4 (Gnbs 1–4) | Quantitative Western blotting of cultured Ric-8A−/− and Ric-8B−/− mouse embryonic stem cell lysates and membranes had substantial reductions in G protein–subunit abundances. Nascent Gαi1/2 and Gαq were defective in initial membrane association and were degraded 10-fold faster in Ric-8A−/− versus WT cells. Hormone-stimulated adenylate cyclase and Rho GTPase activities were reduced in Ric-8–null cells. | Gabay et al. (2011) | ||
| Rabbit reticulocyte lysate | Ric-8A | Immunodepletion and Protein Supplementation | Gαq (GNAQ) Gαq-Q209L Gαi2 (GNAI2) Gα13 (GNA13) | In vitro translated Gα subunits were not folded properly when produced in Ric-8A–immunodepleted RRL. Purified Ric-8A supplementation restored competency of the Ric-8A–depleted RRL to fold functional Gα subunits. | Chan et al. (2013) |
| Xenopus laevis | Ric-8 | Overexpression, siRNA knockdown | Gαs (GNAS) | Microinjection of ric-8 mRNA in Xenopus oocytes potentiated Gαs-inhibition of oocyte maturation. ric-8 siRNA knockdown in primed oocytes caused maturation. | Romo et al. (2008) |
| Caenorhabditis elegans | Ric-8 | Hypomorphic ric-8 mutation, RNAi knockdown | Gαo (goa-1) | Embryos derived from ric-8 and single-copy goa-1 mutant parents were lethal. Centrosome movements were diminished in ric-8 mutants and absent in embryos derived from ric-8/goa-1 parents. | Miller and Rand (2000) |
| Hypomorphic ric-8 mutation | Gαq (egl-30) | ric-8 mutants had similar defects as egl-30 mutants (e.g., aldicarb resistance, reduced locomotion rates). Defects were rescued by treatment with diacylglycerol analogs. | Miller et al. (2000) | ||
| Hypomorphic ric-8 mutations, RNAi knockdown | Gαo (goa-1) Gαi homolog (gpa-16) | ric-8 hypomorphs treated with ric-8 RNAi, had decreased mitotic spindle pole movements, resembling the phenotype of goa-1/gpa-16 RNAi double knockdown embryos. Less Gαo and GPR-1/2 complex was coimmunoprecipitated in ric-8 hypomorphs. | Afshar et al. (2004) | ||
| ric-8–null mutant | Gαq (egl-30) Gαs (gsa-1) | ric-8–null mutants exhibit a phenotype similar to double gsa-1/egl-30 mutants (i.e., paralysis). gsa-1 gain-of-function mutation provided only weak rescue of the ric-8–null mutant phenotype. | Reynolds et al. (2005) | ||
| Hypomorphic ric-8 mutations | Gαi homolog (gpa-16) | ric-8 hypomorphs had decreased overall GPA16 protein levels and reduced plasma membrane localization. | Afshar et al. (2005) | ||
| Drosophila melanogaster | Ric-8 | ric-8–null, RNAi knockdown | Gαi (G-iα65A) Gβ (Gβ13F) | ric-8 mutants had defective Gαi and Gβ plasma membrane localization. Gαi overexpression in ric-8–null cells did not rescue the Gβ membrane localization defect. | David et al. (2005) |
| ric-8–null (maternal and zygotic Ric-8) | GαI (G-iα65A) Gαo (G-oα47A) Gβ (Gβ13F) | Gαi and Gβ protein levels were severely reduced in ric-8 mutant embryos. Gβ no longer coimmunoprecipitated with Gαi from ric-8 mutant cells and embryos. Whole-cell staining showed that Ric-8 was required for plasma membrane localization of Gαi, Gαo, and Gβ. | Hampoelz et al. (2005) | ||
| ric-8–null (maternal and zygotic Ric-8) | Gαi (G-iα65A) Gβ (Gβ13F) | Localization of Gαi and Gβ at the plasma membrane was disrupted in ric-8 mutants. | Wang et al. (2005) | ||
| Insect (Spodoptera frugiperda, Trichopulsia ni) | Mammalian Ric-8A & Ric-8B | Recombinant mammalian baculoviral overexpression | Gαq (GNAQ) Gαi1 (GNAI1) Gα13 (GNA13) Gαs (GNAS) Gαolf (GNAL) | Recombinant Gα coexpression with Ric-8A or B in insect cells boosted Gα levels 25- to 50-fold and permitted an enhanced method to purify recombinant Gα subunits. | Chan et al. (2011a) |
| Dictyostelium discoideum | Ric-8 | ric-8–null mutant gene disruption | Gα2 (gpaB) Gα4 (gpaD) | ric-8 is necessary to maintain abundance of the developmental marker and GPCR cAR1. Proper cAR1 abundance was previously shown to require functional Gα2. Gα4-dependent folate chemotaxis was defective in ric-8–null animals. | Kumagai et al. (1991); Kataria et al. (2013) |
| Neurospora crassa | Ric-8 | ric-8–null mutant gene deletion | GNA-1 (gna-1) GNA-2 (gna-2) GNA-3 (gna-3) GNB-1 (gnb-1) | ric-8–null mutants (Δric8) had reductions of GNA-1, GNA-2, GNA-3, and GNB-1 whole-cell protein levels. G protein transcript levels were not affected. Δric8 mutant and Δgna-1/Δgna-3 double mutant phenotypes were similar (e.g., defects in vegetative growth, absence of female sexual structures, and inappropriate formation of specialized hyphae in submerged cultures). | Wright et al. (2011) |
| ric-8–null mutant gene deletion | GNA-1 (gna-1) GNA-2 (gna-2) GNA-3 (gna-3) | Deletion studies show Ric-8 is required for Gα-dependent development of specialized hyphae. Expression of constitutively active Gα mutants within Δric-8 background was not sufficient to rescue the Δric8 phenotype (e.g., increased proportion of fungal spores). | Eaton et al. (2012) |
COS, CV-1 (simian) in Origin, and carrying the SV40 genetic material cells; ERK, extracellular signal-regulated kinase; MEF, Mouse embryonic fibroblasts; RRL, rabbit reticulocyte lysate; WT, wild type; YFP, yellow fluorescent protein;