Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides. Endogenous 5-HT(2C) receptors in choroid plexus epithelial cells
| Autor: | M, Chang, L, Zhang, J P, Tam, E, Sanders-Bush |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Mitogen-Activated Protein Kinase Kinases
Cell Membrane Permeability Molecular Sequence Data Epithelial Cells Peptide Fragments Rats Rats Sprague-Dawley GTP-Binding Proteins Astrocytes Receptors Serotonin Type C Phospholipases Choroid Plexus Receptor Serotonin 5-HT2C Animals Humans Amino Acid Sequence Virulence Factors Bordetella Cells Cultured |
| Zdroj: | The Journal of biological chemistry. 275(10) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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