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Ric-8 Enhances G Protein -Dependent Signaling in Response to -Binding Peptides in Intact Cells

Departments of Pharmacology and Physiology (S.M., M.G., T.M.B., A.V.S.) and Biochemistry and Biophysics (A.V.S.), University of Rochester School of Medicine and Dentistry, Rochester, New York; and Department of Pharmacology (G.G.T.), University of Texas Southwestern Medical Center, Dallas, Texas

Peptides derived from a random-peptide phage display screen with purified G₁₂ 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 G subunits and accelerating the dissociation of GGDP 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 G₁ subunits (₁W332A) that does not bind SIRK in vitro. Transfection of G₁W332A mutant into Chinese hamster ovary cells blocked peptide-mediated activation of extracellular signal-regulated kinase (ERK), but it did not affect receptor-mediated G subunit-dependent ERK activation, indicating that G subunits are in fact the direct target in cells responsible for ERK activation. To determine whether free G subunits were released from G protein heterotrimers upon peptide treatment, cells were transfected with Ric-8A, a guanine nucleotide exchange factor for free GGDP, 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 G 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 G subunit signaling. Overall, these experiments provide further support for the hypothesis that mSIRK promotes G protein subunit dissociation to release free subunits in intact cells.

Address correspondence to: Dr. Alan V. Smrcka, Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Box 711, Rochester, NY 14642. E-mail: alan_smrcka{at}urmc.rochester.edu

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