A MEMBRANE-PERMEABLE PEPTIDE CONTAINING THE LAST 21 RESIDUES OF THE GAS CARBOXYL TERMINUS INHIBITS GS-COUPLED RECEPTOR SIGNALING IN INTACT CELLS: CORRELATIONS BETWEEN PEPTIDE STRUCTURE AND BIOLOGICAL ACTIVITY

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 Gs. This Gs peptide was an effective inhibitor of 5-N-ethylcarboxamidoadenosine (NECA) and isoproterenolstimulated 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 Gs 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 Gs 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 Gs peptide is a valuable, nontoxic research tool to modulate Gs-coupled receptor signal transduction in cell culture models.