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 alpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [H-3]2-[4-(2-carboxyethyl) phenylethylamino]- 5'-N-ethylcarboxamidoadenosine ([H-3]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The G alpha(s) peptides stimulated specific binding both in the presence 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 presence 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 gamma 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 fitted by one-site models. This same G alpha(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 alpha(s) and G alpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide G alpha(s) (374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of G alpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.
A G alpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor
MAZZONI, MARIA ROSA;GIUSTI, LAURA;LUCACCHINI, ANTONIO;
2000-01-01
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 G alpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [H-3]2-[4-(2-carboxyethyl) phenylethylamino]- 5'-N-ethylcarboxamidoadenosine ([H-3]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The G alpha(s) peptides stimulated specific binding both in the presence 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 presence 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 gamma 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 fitted by one-site models. This same G alpha(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 alpha(s) and G alpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide G alpha(s) (374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of G alpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
