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J Biol Chem, Vol. 275, Issue 10, 7021-7029, March 10, 2000
From the 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
Dissecting G Protein-coupled Receptor Signaling Pathways with
Membrane-permeable Blocking Peptides
ENDOGENOUS 5-HT2C RECEPTORS IN CHOROID PLEXUS
EPITHELIAL CELLS*
,
Department of Pharmacology and Center for
Molecular Neuroscience and the § Department of
Microbiology and Immunology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232
2 adrenergic receptor-mediated activation of cAMP but not
5-HT2C receptor-mediated phosphatidylinositide hydrolysis.
The peptide MPS-PLC1M, mimicking the domain of phospholipase C1
(PLC1) interacting with active G
q, also blocked
5-HT2C receptor activation. In contrast, peptides PLC2M
and Phos that bind to and sequester free G
subunits were
ineffective at blocking 5-HT2C receptor-mediated phosphoinositol turnover. However, both peptides disrupted
G-mediated 2A adrenergic receptor activation of
mitogen-activated protein kinase. These results provide the first
direct demonstration that active Gq subunits mediate
endogenous 5-HT2C receptor activation of PLC and that
G subunits released from Gq 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.
*
This work was supported by National Institutes of Health
research Grants MH34007 (to E. S. B.) and CA36544 (to J. P. T.).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.
To whom correspondence should be addressed. Tel.:
615-936-1685; Fax: 615-343-6532; E-mail:
Elaine.bush@mcmail.vanderbilt.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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