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Originally published In Press as doi:10.1074/jbc.M204753200 on May 29, 2002

J. Biol. Chem., Vol. 277, Issue 32, 28631-28640, August 9, 2002
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Selective Inhibition of Heterotrimeric Gs Signaling
TARGETING THE RECEPTOR-G PROTEIN INTERFACE USING A PEPTIDE MINIGENE ENCODING THE Galpha s CARBOXYL TERMINUS*

David S. FeldmanDagger §, A. Musa Zamah, Kristen L. Pierce, William E. Miller, Francine KellyDagger , Antonio RapacciuoloDagger , Howard A. RockmanDagger , Walter J. Koch||, and Louis M. LuttrellDagger **Dagger Dagger

From the Departments of Dagger  Medicine,  Biochemistry, and || Surgery, Duke University Medical Center, Durham, North Carolina 27710 and the ** Geriatrics Research, Education, and Clinical Center, Durham Veterans Affairs Medical Center, Durham, North Carolina 27705

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha s (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta 2 adrenergic receptors (AR) and inhibited beta 2AR-induced [35S]GTPgamma S loading of Galpha s. GsCT expression inhibited beta 2AR- and dopamine D1A receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-Gs coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgamma S loading of Galpha q/11 by alpha 1BARs and Galpha i by alpha 2AARs and Gq/11- or Gi-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for Gs. We next employed the GsCT construct to examine the complex role of Gs in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta 2AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha 2AAR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the Gq/11-coupled alpha 1BAR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.

* This work was supported by a Howard Hughes Postdoctoral Research Fellowship for Physicians (to D. S. F.) and National Institutes of Health Grant DK55524 (to L. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Division of Cardiology, P. O. Box 250623, Dept. of Medicine, Medical University of South Carolina, Charleston, SC 29425. E-mail: feldmds@musc.edu.

Dagger Dagger To whom correspondence should be addressed: N3019 GRECC, Durham Veterans Affairs Medical Center, 508 Fulton St., Durham, NC 27705. Tel.: 919-286-0411, Ext. 7196; Fax: 919-416-5823; E-mail: luttrell@receptor-biol.duke.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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