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A Membrane-Permeable Peptide Containing the Last 21 Residues of the G_S Carboxyl Terminus Inhibits G_S-Coupled Receptor Signaling in Intact Cells: Correlations between Peptide Structure and Biological Activity

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Pisa, Italy (C.G., L.G., A.L., M.R.M., F.P.); Dipartimento di Scienze Farmaceutiche, Università di Salerno, Fisciano (Sa), Italy (A.M.D., C.E.); Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli "Federico II", Napoli, Italy (S.A., G.C., E.N.); and Laboratorio di Chimica e Biologia di Peptidi e Proteine, Dipartimento di Scienze Farmaceutiche, Università di Firenze, Sesto Fiorentino (Fi), Italy (P.R.)

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_s. This G_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_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_s fragment assumed an amphipathic -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 -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_s peptide is a valuable, nontoxic research tool to modulate G_s-coupled receptor signal transduction in cell culture models.

Received for publication August 5, 2005.

Accepted for publication December 5, 2005.

Address correspondence to: Dr. Maria R. Mazzoni, Dip. di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Via Bonanno 6, 56126 Pisa, Italy. E-mail: mariarm{at}farm.unipi.it

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