A G alpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor

The molecular mechanisms of interaction between G(s) and the A(2A) adenosin e receptor were investigated using synthetic peptides corresponding to vari ous segments of the G alpha(s) carboxyl terminus. Synthetic peptides were t ested for their ability to modulate binding of a selective radiolabeled ago nist, [H-3]2-[4-(2-carboxyethyl) phenylethylamino]- 5'-N-ethylcarboxamidoad enosine ([H-3]CGS21680), to A(2A) adenosine receptors in rat striatal membr anes. The G alpha(s) peptides stimulated specific binding both in the prese nce and absence of 100 mu M guanosine- 5'-O-(3-thiotriphosphate) (GTP gamma S). Three peptides, G alpha(s) (378-394)C(379)A, G alpha(s) (376-394)C(379 )A, and G alpha(s) (374-394)C(379)A, were the most effective. In the presen ce of GTP gamma S, peptide G alpha(s) (374-394) C(379)A 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 [H-3] CGS21680 . Binding assays with a radiolabeled selective antagonist, [H-3]5-amino-7-( 2-henylethyl)-2-(2-furyl) pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ( [H-3]SCH58261), showed that the addition of the G alpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTP g amma S, the displacement curve was right-shifted, whereas the addition of G alpha(s) (374-394)C(379)A caused a partial left-shift. Both curves were fi tted by one-site models. This same G alpha(s) peptide was also able to disr upt G(s)-coupled signal transduction as indicated by inhibition of the A(2A ) receptor-stimulated adenylyl cyclase activity without affecting either ba sal or forskolin-stimulated enzymatic activity in the same membrane prepara tions. Shorter peptides from G alpha(s) and G alpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptid e G alpha(s) (374-394)C(379)A to assume a compact carboxyl-terminal alpha-h elical conformation in solution. Overall, our results point out the conform ation requirement of G alpha(s) carboxyl-terminal peptides to modulate agon ist binding to rat A(2A) adenosine receptors and disrupt signal transductio n.