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J. Biol. Chem. 275 (10): 7021-7029

© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides


Mike Chang{ddagger}, Lianshan Zhang§, James P. Tam§, , and Elaine Sanders-Bush{ddagger}||

From the {ddagger}Department of Pharmacology and Center for Molecular Neuroscience and the §Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

ABSTRACT Back to Top

Abstract: To determine the intracellular signaling mechanism of the 5-HT2C receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains were examined. Only GqCT, which targets the receptor-Gq protein interacting domain, disrupted 5-HT2C receptor-mediated phosphatidylinositide hydrolysis. GsCT, targeting the receptor-Gs protein, disrupted β2 adrenergic receptor-mediated activation of cAMP but not 5-HT2C receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCβ1M, mimicking the domain of phospholipase Cβ1 (PLCβ1) interacting with active Gαq, also blocked 5-HT2C receptor activation. In contrast, peptides PLCβ2M and Phos that bind to and sequester free Gβ{gamma} subunits were ineffective at blocking 5-HT2C receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gβ{gamma}-mediated α2A adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Gαq subunits mediate endogenous 5-HT2C receptor activation of PLCβ and that Gβ{gamma} subunits released from Gαq heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, conjugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.

Received for publication October 27, 1999.

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