Evaluation of the Role of GTP Hydrolysis in the Interaction Between Mg²⁺ and GTP in Regulating Agonist Binding to the Adenosine A₁ Receptor

Abstract

Binding of agonists to adenosine receptors is reduced by GTP, whereas it is enhanced by Mg²⁺. The effect of GTP can be completely reversed by divalent cations, in contrast to the effect of the nonhydrolyzable analogue 5′-guanylylimidodiphosphate (GPPNHP). The present study addresses the role of divalent cation-stimulated specific and nonspecific GTP-ases in this reversal process. Under the conditions commonly employed in binding assays, almost all GTP is rapidly converted to GMP and P_i, indicating that maintenance of GTP levels is essential for the proper interpretation of results. A combination of a GTP-generating system and a competing substrate for high K_m GTP-ases minimizes GTP breakdown. In the presence of these additions, the reversal of GTP effects is almost eliminated, and the inhibitory effects of both GTP and GPPNHP on agonist binding are reduced by divalent cations to a similar extent. Besides enhancing nonspecific GTP hydrolysis, Mg²⁺, but not Mn²⁺ or Ca²⁺, also stimulates specific agonist-dependent GTP-ase activity. Thus, it is evident that specific regulatory effects of Mg²⁺ and other divalent cations can only be identified when other, nonspecific, effects have been evaluated and controlled.