title: Ric-8 enhances G protein betagamma-dependent signaling in response to betagamma-binding peptides in intact cells.
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.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Box 711, Rochester, NY 14642, USA.
authors.forename: Sundeep
authors.initials: S
authors.lastname: Malik
authors.forename: Mousumi
authors.initials: M
authors.lastname: Ghosh
authors.forename: Tabetha M
authors.initials: TM
authors.lastname: Bonacci
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Tall
authors.forename: Alan V
authors.initials: AV
authors.lastname: Smrcka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism* / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D010446:Peptide Fragments; C485885:Ric8a protein, rat