Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides -: Endogenous 5-HT2C receptors in choroid plexus epithelial cells

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 mere examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT2C receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta 2 adrenergic receptor-mediated activation of cAMCP but not 5-HT2C receptor-mediated phosphatidylinositide hydrolysis, The peptide MPS-PLC beta 1M, mimicking the domain of phospholipase C beta 1 (PLC beta 1) interacting with active G alpha(q), also blocked 5-HT2C receptor activation. In contrast, peptides PLC beta 2M and Phos that bind 60 and sequester free G beta gamma subunits were ineffective at blocking 5-HT2C receptor-mediated phosphoinositol turnover. However, both peptides disrupted G beta gamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase, These results provide the first direct demonstration that active G alpha(q) subunits mediate endogenous 5-HT2C receptor activation of PLC beta and that G beta gamma subunits released from G alpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, coqiugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.