Ric-8 enhances G protein beta gamma-dependent signaling in response to beta gamma-binding peptides in intact cells

Department of Pharmacology and Physiology, University of Rochester, Rochester, New York, United States
Molecular Pharmacology (Impact Factor: 4.12). 08/2005; 68(1):129-36. DOI: 10.1124/mol.104.010116
Source: PubMed

ABSTRACT Peptides derived from a random-peptide phage display screen with purified Gbeta(1)gamma(2) subunits as the target promote the dissociation of G protein heterotrimers in vitro and activate G protein signaling in intact cells. In vitro, one of these peptides (SIRKALNILGYPDYD; SIRK) promotes subunit dissociation by binding directly to Gbetagamma subunits and accelerating the dissociation of GalphaGDP without catalyzing nucleotide exchange. The experiments described here were designed to test whether the mechanism of SIRK action in vitro is in fact the mechanism of action in intact cells. We created a mutant of Gbeta(1) subunits (beta(1)W332A) that does not bind SIRK in vitro. Transfection of Gbeta(1)W332A mutant into Chinese hamster ovary cells blocked peptide-mediated activation of extracellular signal-regulated kinase (ERK), but it did not affect receptor-mediated Gbetagamma subunit-dependent ERK activation, indicating that Gbetagamma subunits are in fact the direct target in cells responsible for ERK activation. To determine whether free Galpha subunits were released from G protein heterotrimers upon peptide treatment, cells were transfected with Ric-8A, a guanine nucleotide exchange factor for free GalphaGDP, but not heterotrimeric G proteins. Ric-8A-transfected cells displayed enhanced myristoyl-SIRKALNILGYPDYD (mSIRK)-dependent inositol phosphate (IP) release and ERK activation. Ric-8A also enhanced ERK activation by the G(i)-linked G protein coupled receptor agonist lysophosphatidic acid. Inhibitors of Gbetagamma subunit function blocked Ric-8-enhanced activation of ERK and IP release. These results suggest that one potential function of Ric-8 in cells is to enhance G protein Gbetagamma subunit signaling. Overall, these experiments provide further support for the hypothesis that mSIRK promotes G protein subunit dissociation to release free betagamma subunits in intact cells.

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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 Gα(i), Gα(q), and Gα(13) proteins to <5% of those of wild-type cells. The amounts of Gα(s) and total Gβ protein were partially reduced in Ric-8A(-/-) cells compared to those in wild-type cells, and only the amount of Gα(s) was reduced substantially in Ric-8B(-/-) cells. The abundances of mRNAs encoding the G protein α 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 Gα(i) and Gα(q) were efficiently translated in Ric-8A(-/-) cells but integrated into endomembranes poorly; however, the reduced amounts of G protein α subunits that reached the membrane still bound to nascent Gβγ. Finally, Gα(i), Gα(q), and Gβ(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 Gα subunits with cellular membranes.
    Science Signaling 11/2011; 4(200):ra79. DOI:10.1126/scisignal.2002223 · 7.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Signaling via heterotrimeric G-proteins is evoked by agonist-mediated stimulation of seven transmembrane spanning receptors (GPCRs). During the last decade it has become apparent that Gα subunits can be activated by receptor-independent mechanisms. Ric-8 belongs to a highly conserved protein family that regulates heterotrimeric G-protein function, acting as a non-canonical guanine nucleotide exchange factors (GEF) over a subset of Gα subunits. In this review we discuss the roles of Ric-8 in the regulation of diverse cell functions, emphasizing the contribution of its multiple domain protein structure in these diverse functions.
    Journal of Cellular Biochemistry 09/2012; 113(9):2797-805. DOI:10.1002/jcb.24162 · 3.37 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Immature stage VI Xenopus oocytes are arrested at the G(2)/M border of meiosis I until exposed to progesterone, which induces meiotic resumption through a non-genomic mechanism. One of the earliest events produced by this hormone is inhibition of the plasma membrane enzyme adenylyl cyclase (AC), with the concomitant drop in intracellular cAMP levels and reinitiation of the cell cycle. Recently Gsalpha and Gbetagamma have been shown to play an important role as positive regulators of Xenopus oocyte AC, maintaining the oocyte in the arrested state. However, a question that still remains unanswered, is how the activated state of Gsalpha and Gbetagamma is achieved in the immature oocyte, since no receptor or ligand have been found to be required. Here we provide evidence that xRic-8 can act in vitro and in vivo as a GEF for Gsalpha. Overexpression of xRic-8, through mRNA injection, greatly inhibits progesterone induced oocyte maturation and endogenous xRic-8 mRNA depletion, through siRNA microinjection, induces spontaneous oocyte maturation. These results suggest that xRic-8 is participating in the immature oocyte by keeping Gsalpha-Gbetagamma-AC signaling complex in an activated state and therefore maintaining G2 arrest.
    Journal of Cellular Physiology 03/2008; 214(3):673-80. DOI:10.1002/jcp.21257 · 3.87 Impact Factor