Abstract

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 Gα_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 Gα_s peptides stimulated specific binding both in the presence and absence of 100 μM guanosine-5′-O-(3-thiotriphosphate) (GTPγS). Three peptides, Gα_s(378–394)C³⁷⁹A, Gα_s(376–394)C³⁷⁹A, and Gα_s(374–394)C³⁷⁹A, were the most effective. In the presence of GTPγS, peptide Gα_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 Gα_s peptide modified the slope of the 5′-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPγS, the displacement curve was right-shifted, whereas the addition of Gα_s(374–394)C³⁷⁹A caused a partial left-shift. Both curves were fitted by one-site models. This same Gα_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 Gα_s and Gα_i1/2 carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Gα_s(374–394)C³⁷⁹A to assume a compact carboxyl-terminal α-helical conformation in solution. Overall, our results point out the conformation requirement of Gα_scarboxyl-terminal peptides to modulate agonist binding to rat A_2A adenosine receptors and disrupt signal transduction.