Human A2A adenosine receptors:: High-affinity agonist binding to receptor-G protein complexes containing Gβ4

Agonists bind with higher affinity to G protein-coupled heptahelical receptors than to uncoupled receptors. Recombinant A(1) and A(3) adenosine receptors couple well to G(i/o), but recombinant human A(2A) adenosine receptors (hA(2A)AR) couple poorly to G(s) and bind agonists with K-i values in binding assays that are much higher than ED50 values for functional responses such as coronary dilation and inhibition of neutrophil oxidative burst. In this study, we produced hA(2A)AR-G protein complexes in membranes derived from Sf9 cells quadruply infected with receptors and heterotrimeric G protein subunits. The composition of G(beta) markedly influences coupling such that A(2A)AR-alpha(s)beta(1)gamma(2) are 8+/-2% coupled whereas equivalently expressed A(2A)AR-alpha(s)beta(4)gamma(2) are 40+/-2% coupled. Hence, we were able for the first time to accurately measure high-affinity agonist binding to hA(2A)AR. The agonist 2-[2-(4-amino-3-[I-125]iodophenyl)ethylamino]adenosine binds to coupled and uncoupled hA(2A)AR with K-D values of 0.46 nM and 26 nM, respectively, a difference in affinity of 57-fold. The addition of GTP-gammaS converts all receptors to the low-affinity state. A(2A)AR coupling does not influence binding of antagonists including, I-125-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (I-125-ZM241385), K-D = 0.5 nM. Based on a comparison of high-affinity binding sites, N-6-3-iodo-2-chlorobenzyladenosine-5'-N-methyluronamide is only 8-fold A(3) selective (A(2A) (Ki, H) = 18.3+/-3.2 nM; A(3 Ki, H) = 2.4+/-0.3 nM) and 2-chloro-N-6-cyclopentyladenosine is only 33-fold A(1) selective (A(2A Ki, H) = 11.0+/-1.9; A(1 Ki, H) = 0.3+/-0.1). We conclude that recombinant hA(2A)AR can form a high-affinity receptor-G protein complex with alpha(s)beta(4)gamma(2) that is useful for determining receptor selectivity.