J Biol Chem, Vol. 275, Issue 10, 7021-7029, March 10, 2000

Dissecting G Protein-coupled Receptor Signaling Pathways with Membrane-permeable Blocking Peptides
ENDOGENOUS 5-HT_2C RECEPTORS IN CHOROID PLEXUS EPITHELIAL CELLS^*

Mike Chang, Lianshan Zhang^§¶, James P. Tam^§, and Elaine Sanders-Bush

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

To determine the intracellular signaling mechanism of the 5-HT_2C 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 G_qCT, which targets the receptor-G_q protein interacting domain, disrupted 5-HT_2C receptor-mediated phosphatidylinositide hydrolysis. G_sCT, targeting the receptor-G_s protein, disrupted 2 adrenergic receptor-mediated activation of cAMP but not 5-HT_2C receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLC1M, mimicking the domain of phospholipase C1 (PLC1) interacting with active G_q, also blocked 5-HT_2C receptor activation. In contrast, peptides PLC2M and Phos that bind to and sequester free G subunits were ineffective at blocking 5-HT_2C 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 G_q subunits mediate endogenous 5-HT_2C receptor activation of PLC and that G subunits released from G_q 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.