Received for publication December 7, 2004.
Revised March 24, 2005.
Accepted for publication March 31, 2005.

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

Abstract

Peptides derived from a random-peptide phage display screen with purified G_{1 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 (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 CHO cells blocked peptide mediated activation of ERK but 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 if free G subunits were released from G protein heterotrimers upon peptide treatment, cells were transfected with Ric-8A a GEF for free GGDP but not heterotrimeric G proteins. Ric-8A transfected cells displayed enhanced mSIRK dependent inositol phosphate (IP) release and ERK activation. Ric-8A also enhanced ERK activation by the G_i linked GPCR agonist LPA. 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 G subunits in intact cells.

Key words: Gi family, Phospholipase C's, G protein regulation, Phage display

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