A Membrane-Permeable Peptide Containing the Last 21 Residues of the G S 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 G s. This G s 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 G s for interaction with receptors. The peptide inhibited neither Gqnor 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 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 Gs-coupled receptor signal transduction in cell culture models. G protein-coupled receptors (GPCRs) represent a large family of cell-surface receptors sharing a common transmembrane structure and signal transduction mechanisms. The basic unit of GPCR signaling is composed of a heptahelical receptor, a heterotrimeric GTP-binding protein (G protein), and an effector, such as an enzyme or an anion channel. The binding of an agonist ligand to the GPCR changes its conformation to allow productive coupling with its cognate G protein, leading to the exchange of GTP for GDP on the G subunit and consequent dissociation of G -GTP from the G complex. Multiple sites of interaction cooperate in the physical coupling between the activated receptor and the G protein (Cabrera-Vera et al., 2003). Data from crystallographic, biochemical, and mutagenesis studies indicate that the key This work was supported by Ministero dell’Istruzione, dell’Universitá e della Ricerca grants (to A.L. and M.R.M.) and by a grant from Fondazione Ente Cassa di Risparmio di Firenze (to P.R.). A.M.D. and L.G. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.017715. ABBREVIATIONS: GPCR, G protein-coupled receptor; G s(374–394)C A, a synthetic peptide corresponding to those residues of G s with a cysteine substituted by an alanine; DQF-COSY, double-quantum filter correlation spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser spectroscopy; DPC, dodecyl phosphocholine; NECA, 5 -N-ethylcarboxamidoadenosine; HMEC-1, human microvascular endothelial cell; PBS, phosphate-buffered saline; FBS, fetal bovine serum; F12K, Kaighn’s modified Ham’s F12 medium. 0026-895X/06/6903-727–736$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 3 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 17715/3087360 Mol Pharmacol 69:727–736, 2006 Printed in U.S.A. 727 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from elements of the interaction are primarily the second and third intracellular loops of the receptor making physical contact with the C terminus of the G subunit (Kobilka et al., 1988; Kostenis et al., 1997). The last 50 residues of the G subunits play a central role in discriminating between different receptor subtypes or different functional states of the same receptor (Cabrera-Vera et al., 2003; Havlickova et al., 2003; Slessareva et al., 2003). Pharmacological agents that act as agonists or antagonists of GPCRs are the most common type of drug in clinical use today. Irrespective of their chemical structure, all of these agents have a common mechanism of action in that they act extracellularly either to mimic or to preclude agonist binding to its receptor. As an alternative approach to antagonism of GPCR signaling, the receptor-G protein interface can be targeted with agents that block coupling between the receptor and the G protein intracellularly. Such an approach is expected to produce G protein-specific rather than receptorspecific antagonism. This strategy has produced several successful results using polypeptides derived from either the putative contact surface on the receptor or the G subunit (Freissmuth et al., 1999). In intact cells, membrane-permeable peptides containing the C-terminal sequence of G q and G s disrupt 5-hydroxytryptamine 2c and 2-adrenergic receptor-mediated activation of phospholipase Cand adenylyl cyclase, respectively (Chang et al., 2000). Cellular expression of a 83-residue polypeptide derived from the C terminus of G s inhibits 2adrenergic and dopamine D1A receptor-mediated cAMP production (Feldman et al., 2002). Minigene plasmids encoding oligopeptides representing the last 11 C-terminal residues of G i, G o, G q, G 12, and G 13 have been successfully used to discern the contribution of different G proteins to signaling by M2 muscarinic and thrombin receptors (Gilchrist et al., 1999; Vanhauwe et al., 2002). In a previous article (Mazzoni et al., 2000), we showed that a 21-residue synthetic peptide, G s(374–394)C A, derived from rat G s C terminus inhibits A2A adenosine receptormediated activation of adenylyl cyclase in rat striatal membranes, and it acquires a defined helical conformation in solution. Here, we present both biological and structural data of a membrane-permeable synthetic peptide containing the sequence of the G s(374–394)C A peptide on the C-terminal side. This membrane-permeable peptide was designed using as carrier molecule, penetratin, a 16-residue fragment derived from the homeodomain of the Drosophila melanogaster transcription factor Antennapedia that translocates through biological membranes (Derossi et al., 1994). The membrane-permeable 37-residue peptide (dubbed A42) was able to cross HMEC-1 and PC12 cell plasma membrane inhibiting A2A, A2B adenosine, and -adrenergic receptor-stimulated cAMP production without affecting Gqand Gi-coupled receptor signaling. Structural data indicated the molecular basis of A42 tropism for plasma membrane. According to previous conformational studies of isolated penetratin (Lindberg et al., 2001) and G s fragments (Mazzoni et al., 2000), both A42 segments were arranged in -helical structures so that the presence of one did not affect the conformation and functionality of the other. Our synthetic peptide represents a powerful tool to inhibit Gs-coupled receptor signaling in intact cells. Materials and Methods Adenosine deaminase was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Fetal bovine serum (FBS) was from Cambrex Corporation (East Rutherford, NJ). MCDB 131 medium was purchased from Invitrogen (Carlsbad, CA). Kaighn’s modified Ham’s F12 medium (F12K), penicillin, streptomycin, horse serum, papaverine, 5 -N-ethylcarboxamidoadenosine (NECA), isoproterenol, forskolin, and direct cAMP enzyme immunoassay kit (CA-200) were products of Sigma-Aldrich (St. Louis, MO). 2,4Difluoro-bis(1H-1,2,4–triazol-1ylmethyl)benzyl alcohol (fluconazole) was a product of Pfizer Inc. (New York, NY). All other reagents were from standard commercial sources and were of the highest