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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
- Publication Year :
- 2006
-
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.
- Subjects :
- Magnetic Resonance Spectroscopy
Protein Conformation
Peptide
Adenosine-5'-(N-ethylcarboxamide)
Cell-Penetrating Peptides
PC12 Cells
Adenylyl cyclase
chemistry.chemical_compound
structural studies
MULTIPLE
Cyclic AMP
GTP-Binding Protein alpha Subunits, Gs
POSITION
Internalization
Receptor
Protein secondary structure
media_common
chemistry.chemical_classification
SECONDARY STRUCTURE
Chemistry
peptide
Biochemistry
NMR-SPECTROSCOPY
Molecular Medicine
A(2A) ADENOSINE RECEPTOR
Signal transduction
Signal Transduction
PROTEIN ALPHA-SUBUNITS
ENDOTHELIAL-CELLS
ADENYLYL-CYCLASE
MICELLES
DOMAINS
G-protein
media_common.quotation_subject
Recombinant Fusion Proteins
Endocytosis
Permeability
Structure-Activity Relationship
Animals
Humans
Pharmacology
Cell Membrane
Isoproterenol
Membrane Proteins
Proteins
NMR
Peptide Fragments
Rats
N-terminus
Endothelium, Vascular
Carrier Proteins
Peptides
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....90c0dc6d78fdac0fb6cc6cb0e794642e