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Vol. 61, Issue 2, 455-462, February 2002
4
Departments of Pharmacology (L.J.M.), Medicine (M.A.M., J.L.), and
Chemistry (J.M.R., T.L.M.), University of Virginia, Charlottesville,
Virginia
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 G
markedly influences coupling such that
A2AAR-
s
1
2 are
8 ± 2% coupled whereas equivalently expressed
A2AAR-s42 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 with
KD values of 0.46 nM and 26 nM,
respectively, a difference in affinity of 57-fold. The addition of
GTPS 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 (A2A
Ki, 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 (A2A
Ki, 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
s42 that is useful for
determining receptor selectivity.
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