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

2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês

10.1074/jbc.275.10.7021

1083-351X

Mike Chang, Lianshan Zhang, James P. Tam, Elaine Sanders‐Bush,

Cell Adhesion Molecules Research

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 q CT, which targets the receptor-G q protein interacting domain, disrupted 5-HT 2C receptor-mediated phosphatidylinositide hydrolysis. G s CT, targeting the receptor-G s protein, disrupted β2 adrenergic receptor-mediated activation of cAMP but not 5-HT 2C receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCβ1M, mimicking the domain of phospholipase Cβ1 (PLCβ1) interacting with active Gα q , also blocked 5-HT 2C receptor activation. In contrast, peptides PLCβ2M and Phos that bind to and sequester free Gβγ subunits were ineffective at blocking 5-HT 2C receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gβγ-mediated α 2A adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Gα q subunits mediate endogenous 5-HT 2C receptor activation of PLCβ and that Gβγ subunits released from Gα 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.