RT Journal Article
SR Electronic
T1 A Membrane-Permeable Peptide Containing the Last 21 Residues of the Gα<sub>S</sub> Carboxyl Terminus Inhibits G<sub>S</sub>-Coupled Receptor Signaling in Intact Cells: Correlations between Peptide Structure and Biological Activity
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 727
OP 736
DO 10.1124/mol.105.017715
VO 69
IS 3
A1 Anna Maria D'Ursi
A1 Laura Giusti
A1 Stefania Albrizio
A1 Francesca Porchia
A1 Cinzia Esposito
A1 Gabriella Caliendo
A1 Claudia Gargini
A1 Ettore Novellino
A1 Antonio Lucacchini
A1 Paolo Rovero
A1 Maria Rosa Mazzoni
YR 2006
UL http://molpharm.aspetjournals.org/content/69/3/727.abstract
AB Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of Gαs. This Gαs peptide was an effective inhibitor of 5′-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect. The maximal efficacy of NECA was substantially reduced when PC12 cells were treated with the chimeric peptide, suggesting that it competes with Gαs for interaction with receptors. The peptide inhibited neither Gq- nor Gi-coupled receptor signaling. The use of a carboxy-fluorescein derivative of the peptide proved its ability to cross the plasma membrane of live cells. NMR analysis of the chimeric peptide structure in a membrane-mimicking environment showed that the Gαs fragment assumed an amphipathic α-helical conformation tailored to make contact with key residues on the intracellular side of the receptor. The N-terminal penetratin portion of the molecule also showed an α-helical structure, but hydrophobic and hydrophilic residues formed clustered surfaces at the N terminus and center of the fragment, suggesting their involvement in the mechanism of penetratin internalization by endocytosis. Our biological data supported by NMR analysis indicate that the membrane-permeable Gαs peptide is a valuable, nontoxic research tool to modulate Gs-coupled receptor signal transduction in cell culture models.