IDENTIFICATION OF DOMAINS OF THE HUMAN A(1) ADENOSINE RECEPTOR THAT ARE IMPORTANT FOR BINDING-RECEPTOR SUBTYPE-SELECTIVE LIGANDS USING CHIMERIC A(1)/A(2A) ADENOSINE RECEPTORS

To provide new insights into the regions of the human A(1) adenosine receptor (A(1)AR) involved in ligand binding, a series of chimeric human A(1) and rat A(2a) adenosine receptors (A(1)/A(2a)) were studied. Binding studies were initially performed on acutely transfected COS cells using fixed doses of the A(2a)AR agonist [H-3]CGS-21680, the A(1)AR agonist [H-3]2-chloro-N-6-cyclopentyladenosine (CCPA), and the A(1)AR antagonist [H-3]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the region of the A(2a)AR from the amino terminus to the end of transmembrane (TM) 1 was replaced by the corresponding region of the A(1)AR (A(1)TM1/A(2a)), [H-3]CGS-21680 and [H-3]CCPA binding was detectable. When an A(1)TM1-2/A(2a) construct was studied, [H-3]CGS-21680 binding was lost and [H-3] DPCPX binding appeared. Saturation studies using [H-3]CCPA revealed that the A(1)TM1/A(2a) construct had low affinity. However, with the subsequent addition of A(1)AR TMs 2-4 receptor affinity improved markedly. Saturation studies using [H-3]DPCPX also revealed that the TMs 1-4 of the A(1)AR conferred wild-type receptor affinity. When the ligand binding properties of A(1)TM1-4/A(2a), A(1)TM1-6/A(2a), and wild type A(1)AR constructs were directly compared, no differences were found using 10 different compounds. When truncated A(1)ARs that extended from the amino terminus to shortly after TM4 were examined, no binding was detectable suggesting that the amino half of the receptor alone is not sufficient for ligand binding. Collectively, these data suggest that the important determinants for A(1)AR agonist and antagonist binding and ligand specificity are present in TMs 1-4.