ROLE OF CYSTEINE RESIDUES OF RAT A(2A) ADENOSINE RECEPTORS IN AGONISTBINDING

In the present study, we investigated the role of disulfide bridges an d sulfhydryl groups in A(2a) adenosine receptor binding of the agonist l)phenylethylamino)-5'-N-ethylcarboxamidoadenosine (CGS 21680). To ev aluate the presence of essential disulfide bridges, rat striatal membr anes were incubated with [H-3]CGS 21680 in the presence of dithiothrei tol and binding of the agonist to membranes was measured. The amount o f [H-3]CGS 21680 which specifically bound, decreased progressively upo n pretreatment of membranes with increasing concentrations of dithioth reitol, Pretreatment of rat striatal membranes with 12.5 mM dithiothre itol for 15 min at 25 degrees C resulted in a 2-fold decrease of A(2a) adenosine receptor affinity for [3H]CGS 21680, and a reduction in the maximal number of binding sites. The presence of agonist or antagonis t ligands protected the A(2a) adenosine receptor sites from the effect of dithiothreitol. We also examined the susceptibility of A(2a) adeno sine receptors to inactivation by the sulfhydryl alkylating reagent, N -ethylmaleimide. When rat striatal membranes were pretreated with N-et hylmaleimide for 30 minutes at 37 degrees C, a decrease in specific [H -3]CGS 21680 binding was observed. Pretreatment of membranes with 1 mM N-ethylmaleimide also resulted in a 2-fold reduction of A(2a) adenosi ne receptor affinity for [H-3]CGS 21680, as well as a slight decrease in the maximal number of binding sites. Neither agonist nor antagonist ligands were effective in protecting the receptor sites from inactiva tion by N-ethylmaleimide. protecting the receptor sites from inactivat ion by N-ethylmaleimide. This protective effect was significant but no t complete. Our data suggest that disulfide bridges play a role in the structural integrity of the A(2a) adenosine receptor, furthermore, re duced sulfhydryl groups appear to be important but we do not yet know if they are on the receptor or on the G(s alpha) subunit.