title: 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.
abstract: 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.
authors.affiliation: Dip. di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: D'Ursi
authors.forename: Laura
authors.initials: L
authors.lastname: Giusti
authors.forename: Stefania
authors.initials: S
authors.lastname: Albrizio
authors.forename: Francesca
authors.initials: F
authors.lastname: Porchia
authors.forename: Cinzia
authors.initials: C
authors.lastname: Esposito
authors.forename: Gabriella
authors.initials: G
authors.lastname: Caliendo
authors.forename: Claudia
authors.initials: C
authors.lastname: Gargini
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.forename: Antonio
authors.initials: A
authors.lastname: Lucacchini
authors.forename: Paolo
authors.initials: P
authors.lastname: Rovero
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: Mazzoni
mesh_terms: D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000037:antagonists & inhibitors*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D057846:Cell-Penetrating Peptides; D000242:Cyclic AMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010455:Peptides; D010539:Permeability; D011487:Protein Conformation; D011506:Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; C509549:peptide A42; D019830:Adenosine-5'-(N-ethylcarboxamide); C414409:penetratin; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol