Vol. 58, Issue 1, 226-236, July 2000

A G_s Carboxyl-Terminal Peptide Prevents G_s Activation by the A_2A Adenosine Receptor

Maria R. Mazzoni, Simone Taddei, Laura Giusti, Paolo Rovero, Claudia Galoppini, Annamaria D'Ursi, Stefania Albrizio, Antonio Triolo, Ettore Novellino, Giovanni Greco, Antonio Lucacchini, and Heidi E. Hamm

Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Pisa, Italy (M.R.M., S.T., L.G., A.L.); Dipartimento di Scienze Farmaceutiche, Università di Salerno, Salerno, Italy (P.R., A.D., S.A.); Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli "Federico II", Napoli, Italy (E.N., G.G.); Istituto di Mutagenesi e Differenziamento, Peptide Synthesis Lab, Consiglio Nazionale delle Ricerche, Pisa, Italy (C.G.); Menarini Ricerche, Firenze, Italy (A.T.); and Northwestern University Institute for Neuroscience, Department of Molecular Pharmacology and Biological Chemistry, Chicago, Illinois (H.E.H.)

The molecular mechanisms of interaction between G_s and the A_2A adenosine receptor were investigated using synthetic peptides corresponding to various segments of the G_s carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [³H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([³H]CGS21680), to A_2A adenosine receptors in rat striatal membranes. The G_s peptides stimulated specific binding both in the presence and absence of 100 µM guanosine-5'-O-(3-thiotriphosphate) (GTPS). Three peptides, G_s(378-394)C³⁷⁹A, G_s(376-394)C³⁷⁹A, and G_s(374-394)C³⁷⁹A, were the most effective. In the presence of GTPS, peptide G_s(374-394)C³⁷⁹A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A_2A adenosine receptor for [³H]CGS21680. Binding assays with a radiolabeled selective antagonist, [³H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([³H]SCH58261), showed that the addition of the G_s peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPS, the displacement curve was right-shifted, whereas the addition of G_s(374-394)C³⁷⁹A caused a partial left-shift. Both curves were fitted by one-site models. This same G_s peptide was also able to disrupt G_s-coupled signal transduction as indicated by inhibition of the A_2A receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from G_s and G_i1/2 carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide G_s(374-394)C³⁷⁹A to assume a compact carboxyl-terminal -helical conformation in solution. Overall, our results point out the conformation requirement of G_s carboxyl-terminal peptides to modulate agonist binding to rat A_2A adenosine receptors and disrupt signal transduction.