A Membrane-Permeable Peptide Containing the Last 21 Residues of the Gα_SCarboxyl 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␣ s . This G␣ s peptide was an effective inhibitor of 5Ј-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 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.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 ABBREVIATIONS: GPCR, G protein-coupled receptor; G␣ s (374 -394)C 379 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, 5Ј-N-ethylcarboxamidoadenosine; HMEC-1, human microvascular endothelial cell; PBS, phosphate-buffered saline; FBS, fetal bovine serum; F12K, Kaighn's modified Ham's F12 medium.

Signal Protein-Derived Peptides as Functional Probes and Regulators of Intracellular Signaling

“…It was shown that peptides, derivatives of C-terminal sites of the G α s , very different in length disturb by the competitive mechanism the functional interaction of receptors and G s proteins and change the affinity of receptors for hormones [66, 77, 143–151]. The expression of 83-mer polypeptide derived from C-terminal region of the G α s induces the inhibition of β 2 -AR- and D 1A -DR-mediated cAMP production in HEK293 cells [146].…”

“…A 37-mer peptidic constrain (A42) containing a 16-mer membrane-permeable sequence (MPS) of penetratin, derived from the homeodomain of the Drosophila melanogaster transcription factor Antennapedia that translocates through membranes, and a 21-mer peptide 374–394(C 379 A) was designed and shown to have no affect on cell viability but to significantly inhibit adenosine receptor-mediated cAMP accumulation in PC12 cells with an EC 50 of 5  μ M and decrease A 2A -adenosine receptor- and β -AR-mediated cAMP production in human cell line HMEC-1 [151]. The maximal efficacy of NECA, a selective agonist of A 2A -adenosine receptor, is substantially reduced in the presence of A42 peptide suggesting that it competes with G α s for the interaction with receptor.…”

“…Using NMR analysis it was shown that A42 peptide is arranged in two stretches of α -helical structure encompassing sequences 3–10 and 18–36. Even in a membrane-mimicking environment and in conjugation with MPS, the segment 18–36 has the secondary structure resembling those of peptide 374–394(C 379 A) and of C-terminal region in full-size G α s [151]. The helix 18–36 is amphipathic and has a polar surface lined by Asp 378 , Arg 390 , Asp 381 , and Arg 385 and two hydrophobic surfaces formed by less polar residues Ile 382 , Ile 383 , Met 386 , His 387 , Tyr 391 , and Leu 393 .…”

Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line, NCI-H28, with Homozygous Deletion of the β-Catenin Gene

“…The non-selective PAR 1 -AP (SFLLRN-NH 2 ) was synthesized in Dr. A.M. D’Ursi’s laboratory (Department of Pharmacy, University of Salerno, Fisciano, Italy) using a conventional solid-phase strategy based on the Fmoc/t-Bu protection chemistry as previously described [30]. Human α-thrombin (high activity, ≥2,800 NIH U/mg protein) was a product of Calbiochem (EMD Millipore Biosciences, Billerica, MA, USA).…”

A perspective on more effective GPCR-targeted drug discovery efforts

“…A single amino-acid substitution (Arg389Pro) in the carboxyl terminus of G α s renders it unresponsive to β 2 adrenergic receptors, and antibodies recognizing G α carboxy-terminal domains block receptor-G protein interactions [114] . Sequence-specific carboxy-terminal peptides [115][116][117] and cell-penetrating peptides [118] can serve as competitive inhibitors for the receptor-G protein interaction. Finally, several mutational assays indicate that the carboxyl terminus of G α confers receptor selectivity [119][120][121][122] .…”