Human A2AAdenosine Receptors: High-Affinity Agonist Binding to Receptor-G Protein Complexes Containing Gβ4

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Abstract
Agonists bind with higher affinity to G protein-coupled heptahelical receptors than to uncoupled receptors. Recombinant A1 and A3 adenosine receptors couple well to Gi/o, but recombinant human A2A adenosine receptors (hA2AAR) couple poorly to Gs and bind agonists with Ki 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 hA2AAR-G protein complexes in membranes derived from Sf9 cells quadruply infected with receptors and heterotrimeric G protein subunits. The composition of Gβ markedly influences coupling such that A2AAR-αsβ1γ2 are 8 ± 2% coupled whereas equivalently expressed A2AAR-αsβ4γ2 are 40 ± 2% coupled. Hence, we were able for the first time to accurately measure high-affinity agonist binding to hA2AAR. The agonist 2-[2-(4-amino-3-[125I]iodophenyl)ethylamino]adenosine binds to coupled and uncoupled hA2AAR withKD values of 0.46 nM and 26 nM, respectively, a difference in affinity of 57-fold. The addition of GTPγS converts all receptors to the low-affinity state. A2AAR coupling does not influence binding of antagonists including,125I-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (125I-ZM241385), KD = 0.5 nM. Based on a comparison of high-affinity binding sites,N6-3-iodo-2-chlorobenzyladenosine-5′-N-methyluronamide is only 8-fold A3 selective (A2AKi, H = 18.3 ± 3.2 nM; A3 Ki, H = 2.4 ± 0.3 nM) and 2-chloro-N6-cyclopentyladenosine is only 33-fold A1 selective (A2AKi, H = 11.0 ± 1.9; A1 Ki, H = 0.3 ± 0.1). We conclude that recombinant hA2AAR can form a high-affinity receptor-G protein complex with αsβ4γ2 that is useful for determining receptor selectivity.

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