Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells.
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 beta2 adrenergic receptor-mediated activation of cAMP but not 5-HT(2C) receptor-mediated phosphatidylinositide hydrolysis. The peptide MPS-PLCbeta1M, mimicking the domain of phospholipase Cbeta1 (PLCbeta1) interacting with active Galpha(q), also blocked 5-HT(2C) receptor activation. In contrast, peptides PLCbeta2M and Phos that bind to and sequester free Gbetagamma subunits were ineffective at blocking 5-HT(2C) receptor-mediated phosphoinositol turnover. However, both peptides disrupted Gbetagamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase. These results provide the first direct demonstration that active Galpha(q) subunits mediate endogenous 5-HT(2C) receptor activation of PLCbeta and that Gbetagamma subunits released from Galpha(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.
Chang
M
M
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Zhang
L
L
Tam
J P
JP
Sanders-Bush
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002831:Choroid Plexus / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D044405:Receptor, Serotonin, 5-HT2C; D011985:Receptors, Serotonin / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D010446:Peptide Fragments; D044405:Receptor, Serotonin, 5-HT2C; D011985:Receptors, Serotonin; D010566:Virulence Factors, Bordetella; D020929:Mitogen-Activated Protein Kinase Kinases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins