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

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2013; 110(10):3794-9. DOI: 10.1073/pnas.1220943110
Source: PubMed


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-AlF-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-AlF-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.

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    • "RIC8 is highly conserved protein required for the mitotic spindle orientation and asymmetric cell division in the early embryogenesis in C. elegans and in D. melanogaster and in mammalian cells (Miller and Rand, 2000; Miller et al., 2000; David et al., 2005; Wang et al., 2005; Woodard et al., 2010). Recent studies have revealed an additional function for RIC8A as a molecular chaperone that regulates G-protein biosynthesis (Gabay et al., 2011; Thomas et al., 2011; Chan et al., 2013), and the importance of RIC8A in adhesion and in migration (Wang et al., 2011; Ma et al., 2012; Fuentealba et al., 2013). Nonetheless, little is known about the function of RIC8A in mammalian development. "
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    ABSTRACT: RIC8A is a non-canonical guanine nucleotide exchange factor (GEF) for a subset of Gα subunits. RIC8A has been reported in different model organisms to participate in the control of mitotic cell division, cell signalling, development and cell migration. Still, the function of RIC8A in the mammalian nervous system has not been sufficiently analysed yet. Adult mice express RIC8A in the brain regions involved in the regulation of memory and emotional behaviour.To elucidate the role of RIC8A in mammalian neurogenesis we have inactivated Ric8a in neural precursor cells by using Cre/Lox system. As a result, the conditional knockout mice were born at expected Mendelian ratio, but died or were cannibalized by their mother within 12 h after birth. The cerebral cortex of the newborn Nes;Ric8aCKO> mice was thinner compared to littermates and the basement membrane was discontinuous, enabling migrating neurons to invade to the marginal zone. In addition, the balance between the planar and oblique cell divisions was altered, influencing the neuron production. Taken together, RIC8A has an essential role in the development of mammalian nervous system by maintaining the integrity of pial basement membrane and modulating cell division. This article is protected by copyright. All rights reserved.
    Developmental Neurobiology 08/2015; 75(9). DOI:10.1002/dneu.22264 · 3.37 Impact Factor
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    • "Ric8 is a long domain that acts as a GEF, activating G alpha subunits in the absence of GPCR signaling, or as a chaperone to stabilize G alpha (Hinrichs et al. 2012; Chan et al. 2013). Ric8-mediated activation of monomeric G alpha is involved in development and signaling in metazoans, fungi, and Dictyostelium (Hinrichs et al. 2012; Kataria et al. 2013). "
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    ABSTRACT: The G Protein Coupled Receptor (GPCR) signalling system is one of the main signalling pathways in eukaryotes. Here we analyse the evolutionary history of all its components, from receptors to regulators, to gain a broad picture of its system-level evolution. Using eukaryotic genomes covering most lineages sampled to date, we find that the various components of the GPCR signalling pathway evolved independently, highlighting the modular nature of this system. Our data show that some GPCR families, G proteins and Regulators of G proteins (RGS) diversified through lineage-specific diversifications and recurrent domain shuffling. Moreover, most of the gene families involved in the GPCR signalling system were already present in the Last Common Ancestor of Eukaryotes (LECA). Furthermore, we show that the unicellular ancestor of Metazoa already had most of the cytoplasmic components of the GPCR signalling system, including, remarkably, all of the G protein alpha subunits, which are typical of metazoans. Thus, we show how the transition to multicellularity involved conservation of the signalling transduction machinery, as well as a burst of receptor diversification to cope with the new multicellular necessities.
    Genome Biology and Evolution 02/2014; 6(3). DOI:10.1093/gbe/evu038 · 4.23 Impact Factor
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    ABSTRACT: 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.
    Journal of Receptor and Signal Transduction Research 02/2013; 33(3). DOI:10.3109/10799893.2013.763828 · 2.28 Impact Factor
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