A membrane-permeable peptide containing the last 21 residues of the G alpha(s) carboxyl terminus inhibits G(s)-coupled receptor signaling in intact cells: correlations between peptide structure and biological activity

• Published in: Molecular pharmacology Vol. 69; no. 3; p. 727
• Main Authors: D'Ursi, Anna Maria, Giusti, Laura, Albrizio, Stefania, Porchia, Francesca, Esposito, Cinzia, Caliendo, Gabriella, Gargini, Claudia, Novellino, Ettore, Lucacchini, Antonio, Rovero, Paolo, Mazzoni, Maria Rosa
• Format: Journal Article
• Language: English
• Published: United States 01-03-2006
• Subjects: Adenosine-5'-(N-ethylcarboxamide) - antagonists & inhibitors; Animals; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Membrane - metabolism; Cyclic AMP - metabolism; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; GTP-Binding Protein alpha Subunits, Gs - antagonists & inhibitors; GTP-Binding Protein alpha Subunits, Gs - chemistry; GTP-Binding Protein alpha Subunits, Gs - metabolism; Humans; Isoproterenol - pharmacology; Magnetic Resonance Spectroscopy; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membrane Proteins - pharmacology; PC12 Cells; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Permeability; Protein Conformation; Proteins; Rats; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Signal Transduction - drug effects; Structure-Activity Relationship
• ISSN: 0026-895X
• DOI: 10.1124/mol.105.017715

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 G alpha(s). This G alpha(s) 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 G alpha(s) for interaction with receptors. The peptide inhibited neither G(q)- nor G(i)-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 G alpha(s) fragment assumed an amphipathic alpha-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 alpha-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 G alpha(s) peptide is a valuable, nontoxic research tool to modulate G(s)-coupled receptor signal transduction in cell culture models.